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The Role of Free Radicals in Chronic Rhinosinusitis
Aaron D. Friedman, BA;
Jay B. Shah, BA;
Thomas G. Takoudes, MD;
Joseph Haddad, Jr, MD
Arch Otolaryngol Head Neck Surg. 2002;128:1055-1057.
ABSTRACT
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Objective To determine whether there is an increased amount of free radical–mediated
damage in diseased vs healthy tissue from patients with chronic rhinosinusitis.
Design Pathophysiologic study. Samples of heathly and diseased tissue were
taken from each patient. Lipid peroxides (LPOs) are a by-product of free radical–mediated
damage; LPO levels and LPO/protein ratios were determined for each patient.
Subjects Consecutive series of 13 human subjects undergoing functional endoscopic
sinus surgery to treat chronic rhinosinusitis.
Results The mean LPO/protein ratio for healthy tissue was 3.52 x 10-5, while that for the diseased tissue was 3.49 x 10-5.
There was no statistically significant difference in the LPO/protein ratio
between healthy and diseased tissue (95% confidence interval, -3.00
x 10-5 to 2.94 x 10-5).
Conclusion Free radical–induced damage, if present, was the same in infected
and control tissues in this pilot investigation into the pathophysiologic
characteristics of human chronic rhinosinusitis.
INTRODUCTION
FREE RADICALS are highly reactive species containing 1 or more unpaired
electrons. They are produced in vivo during normal metabolism by enzymes such
as xanthine oxidase and nitric oxide synthase and most abundantly by the electron
transport chain during oxidative phosphorylation. Polymorphonuclear leukocytes
also generate free radicals as part of the inflammatory response. Although
transient, species such as the superoxide radical (O2·-) and the hydroxyl radical (OH·) can overwhelm natural
antioxidant defenses, altering proteins, nucleic acids, and lipids (lipid
peroxidation). This can result in cell injury or death, subsequent tissue
damage, and ultimately, a chronic disease state.1
Free radical–mediated damage has been characterized in the pathogenesis
of more than 100 disorders including stroke, atherosclerosis, myocardial infarction,
autoimmune diseases, and nervous system disorders.1
Studies in our laboratory have implicated free radicals in otitis media (OM)
in animal models2-5
and humans.6 Neutrophils, in their respiratory
burst, and Streptococcus pneumoniae, the most common
pathogen in acute OM,7 are thought to generate
the damaging species in this infection.
The previous findings that free radicals are indeed involved in OM prompt
a search for them in other related diseases. Although clinically separate,
rhinosinusitis and OM share a common pathogenesis. Both are closed-space infections
involving the blockage of a natural orifice (the eustachian tube in OM and
the sinus ostia in rhinosinusitis) leading to stasis and subsequent bacterial
infection.
No recent studies in the English-language literature have assessed for
free radical–mediated damage in human rhinosinusitis. Tissue samples
from patients undergoing functional endoscopic sinus surgery to treat chronic
rhinosinusitis were collected and the lipid peroxide (LPO) content of control
and diseased mucosa was determined.
SUBJECTS AND METHODS
A series of 13 consecutive patients recruited over a 10-week period
underwent functional endoscopic sinus surgery to treat chronic rhinosinusitis.
All patients signed a standard informed consent form allowing for research
evaluation of surgical tissues. None of the patients were taking systemic
steroids prior to surgery. Samples of the diseased mucosa (chosen for obvious
signs of inflammatory thickening and erythema) and corresponding control samples
from healthy-appearing mucosa in the nasal septum, inferior turbinate, or
ethmoid sinus were obtained for each case. Because the study dealt with removed
tissue and did not affect the patients themselves, the institutional review
board granted exemption from the formal approval process.
The tissues were placed in a solution containing cold 0.1M triethanolamine-buffered
saline with 1mM phenylmethylsulfonyl fluoride, 1mM EDTA, and 1mM dithioerythritol.
Samples were mechanically homogenized with a glass grinder, disrupted with
a sonicator for 3 seconds, and centrifuged at 1000g
for 5 minutes at 4°C. The supernatant was frozen in a light-deprived environment
until all samples were collected and ready to be analyzed. Two separate aliquots
from each of the control and diseased supernatants were processed. Sample
protein concentration was determined colorimetrically with the BCA protein
assay (Pierce Endogen, Rockford, Ill) as described by Smith et al.8
Because molecules such as the superoxide and hydroxyl radicals are transient
species that are difficult to measure directly, the amount of LPO, a by-product
of free radical damage to cell membranes, was quantified. The Determiner LPO-CC
kit (Kamiya Biomedical, Thousand Oaks, Calif) uses the hemoglobin-catalyzed
stoichiometric reaction of hydroperoxides with 10-N-methylcarbamoyl-3,7-dimethylamino-10-H-phenotholthiazine
(MCDP) to form methylene blue, which can be measured with a spectrophotometer.
In a light-deprived environment, samples were mixed with kit reagent
1 (ascorbic oxidase and lipoprotein lipase) and incubated at 37°C for
10 minutes. Kit reagent 2 (MCDP) was then added and the samples were reincubated
at 37°C for 15 minutes. Absorbance was measured at 675 nm using a microplate
reader, and the LPO content (expressed in nanomoles per milliliter) was determined
relative to a known cumene standard provided with the kit. Each of the 2 aliquots
from the control and diseased groups was processed in triplicate. Therefore,
a total of 12 LPO concentrations were obtained (6 for the control and 6 for
the diseased tissue) for each patient. Mean control and diseased values were
then divided by their respective sample protein concentrations to yield LPO/protein
ratios for each patient. A paired t test with 12 df was used to ascertain whether a statistically significant
difference in free radical–mediated damage between normal and chronically
inflamed tissue was present.
RESULTS
Figure 1 depicts the LPO/protein
ratios from control and diseased tissue samples for each of the 13 patients.
There was not a statistically significant difference in the degree of free
radical–mediated damage between healthy and inflamed tissue (95% confidence
interval, -3.00 x 10-5 to 2.94 x 10-5).
The mean LPO/protein ratio for healthy tissue was 3.52 x 10-5
with an SE of 1.57 x 10-7, while that for the diseased tissue
was 3.49 x 10-5 with an SE of 1.73 x 10-5
(Figure 2).
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Figure 1. Lipid peroxide (LPO)/protein ratios
of mucosal samples from patients undergoing functional endoscopic sinus surgery
to treat chronic rhinosinusitis; LPO/protein ratios are markers of free radical
damage.
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Figure 2. Mean lipid peroxide (LPO)/protein
ratios of mucosal samples from 13 patients undergoing functional endoscopic
sinus surgery to treat chronic rhinosinusitis; LPO/protein ratios are markers
of free radical damage. Bars indicate SE.
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Biopsy specimens of the nasal turbinates were taken as controls in most
patients (10/13) while the ethmoid sinus was the most frequent biopsy site
for diseased tissue (10/13). One patient (No. 3) had diseased and control
biopsy specimens taken from inflamed and healthy areas (as determined by the
operating surgeon), respectively, of the ethmoid sinus.
COMMENT
The data obtained are the result of a pilot study of free radical–mediated
damage in human patients with chronic rhinosinusitis. There seems to be no
difference between healthy and inflamed tissue. This may be due to the chronicity
of disease in the study patients. Previous work in our laboratory has demonstrated
that guinea pigs with experimentally induced OM have statistically significant
decreases in lipoperoxidation at 30 days after infection vs 5 days after infection4 and that levels of hydrogen peroxide, a by-product
of oxidative metabolism and significant mediator of free radical–induced
damage, are highest 24 hours after infection.3
All 13 patients in the present study had chronic rhinosinusitis, which, by
definition, necessitates a symptom duration of 12 weeks or longer.9 Perhaps free radical–mediated damage in rhinosinusitis
wanes with time because antioxidant defense mechanisms, which are overwhelmed
in the acute stage of disease, catch up as inflammation persists and time
allows for tissue repair. Moreover, the nature of the inflammatory response
is known to be different in acute vs chronic disease. Acute sinusitis leads
to a substantial influx of neutrophils,10 which
generate free radicals during their respiratory burst; however, studies evaluating
the histopathologic characteristics of chronic sinusitis have shown a predominantly
lymphocytic response, with only occasional neutrophils present.11-12
Thus, lipoperoxidation may have occurred initially, during the acute phase
of rhinosinusitis, but not during the chronic inflammation that the study
subjects were known to endure. As mucosal tissue healed from the initial insult,
perhaps free radical–induced damage decreased as well.
Two possible experimental design aspects may also account for the lack
of difference in lipoperoxidation. First, the use of a control specimen from
the same patients with diseased tissue eliminated the possibility of confounding
but also relied on the surgeon's ability to choose healthy tissue based on
visual inspection. Given the chronicity of the patients' disease, it is certainly
possible that tissue that did not appear inflamed at the time of surgery was
affected prior to the procedure and may have suffered free–radical mediated
damage that was subsequently detected by the LPO assay. This raises the possibility
that control samples were not representative of healthy tissue.
Second, tissue samples were initially processed and frozen until further
analysis was possible. The freezing process can generate radicals capable
of damaging biological molecules and, in fact, this is a common problem in
the production of proteins for biological use.1
The prolonged freezing of the samples had an uncertain effect on final results.
It is important to note, however, that freezing was also used in the storage
of samples during previous experiments in our laboratory, experiments that
did demonstrate a difference in free radical–mediated damage.2-6
Although this study showed no statistically significant difference in
LPO content between healthy and diseased tissue in chronic rhinosinusitis,
perhaps the difference, if it exists at all, is smaller and therefore more
difficult to detect than that which has been shown in OM. While the neutrophil
respiratory burst is a part of the inflammatory response and contributes to
free radical formation in OM and rhinosinusitis, S pneumoniae, the other source of free radicals in OM, may not play as large a
role in chronic rhinosinusitis. Though it is one of the most common pathogens
in acute sinusitis, pneumococcus is relatively rare in cultures from patients
with chronic sinusitis. A recent study of 174 patients with chronic maxillary
sinusitis grew S pneumoniae in only 0.5% of cultures,
and a review of the literature failed to implicate it as a predominant organism
in 7 of the 9 most recently published articles on the subject.13
In conclusion, the present data represent a preliminary investigation
into the role of free radicals in chronic rhinosinusitis. Further studies
involving larger numbers of samples and the use of an animal model for rhinosinusitis
are planned.
AUTHOR INFORMATION
Accepted for publication February 27, 2002.
Corresponding author: Joseph Haddad, Jr, MD, Columbia-Presbyterian
Medical Center, Babies' and Children's Hospital, 3959 Broadway, Room 501N,
New York, NY 10032 (e-mail: jh56{at}columbia.edu).
From the Department of Otolaryngology–Head and Neck Surgery,
New York Presbyterian Hospital and College of Physicians and Surgeons, Columbia
University, New York.
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