Acta Oto-Laryngologica
ISSN: 0001-6489 (Print) 1651-2251 (Online) Journal homepage: http://www.tandfonline.com/loi/ioto20
Superoxide Dismutase in Middle Ear Fluid from Children with Secretory Otitis Media Therese Ovesen & Jens D. Börglum To cite this article: Therese Ovesen & Jens D. Börglum (1992) Superoxide Dismutase in Middle Ear Fluid from Children with Secretory Otitis Media, Acta Oto-Laryngologica, 112:6, 1017-1024, DOI: 10.3109/00016489209137504 To link to this article: http://dx.doi.org/10.3109/00016489209137504
Published online: 08 Jul 2009.
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Citing articles: 1 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ioto20 Download by: [University of California Santa Barbara]
Date: 05 May 2016, At: 18:49
Acta Otolaryngol (Stockh) 1992; 112: 1017-1024
Superoxide Dismutase in Middle Ear Fluid from Children with Secretory Otitis Media THERESE OVESEN' and JENS D. BORGLUM*
Downloaded by [University of California Santa Barbara] at 18:49 05 May 2016
From the 'ENT Department, University Hospital of Aarhus, and 'University Clinic of Endocrinology and Internal Medicine, Aarhus County Hospital, Aarhus, Denmark
Ovensen T, Borglum JD. Superoxide dismutase in middle ear fluid from children with secretory otitis media. Acta Otolaryngol (Stockh) 1992; 1 1 2 1017-1024. Superoxide dismutase (SOD) constitutes an important enzymatic defense against the destructive biochemical events associated with inflammation. To examine the presence of SOD in the middle ear fluid (MEF) in secretory otitis media (SOM) the content of SOD was measured in the MEF from 66 children with SOM. The applied method was based on light emission associated with reduction of the chemiluminescence of luminol reacting with superoxide anions (OF). From the inhibition exerted by the addition of standard SOD the concentration of SOD in the samples was extrapolated. The findings divided the MEF's into two significantly different groups: 48% with undetectable SOD, 52% with a SOD range of 200-12,000 ng/ml, median: 3,333 ng/ml, 95% confidence limits: 2,000-5,ooO ngjml ( p < 0.0005). It is suggested that the concentration of superoxide dismutase in the middle ear may play an essential role for the outcome of secretory otitis media and the effect of insertion of ventilation tubes. Key words: superoxide dismutase, secretory otitis media.
INTRODUCTION Secretory otitis media (SOM) is an inflammatory middle ear disease characterized by secretory transformation of the epithelium lining the middle ear cavity and subepithelial oedema, infiltration of phagocytes, vascular proliferation and hypertrophia/hyperplasia of the connective tissue ( 1-4). Furthermore, SOM is accompanied by fluid in the cavity, which may result from either transudation or exudation and active secretion from the epithelial cells or a combination of these processes ( 1-4). Etiologically and pathogenetically both dysfunction of the Eustachian tube resulting in a negative middle ear pressure and infectious spreading from the nasopharynx seem to be involved (3, 4, 5). The clinical picture ranges from transient, spontaneously healing episodes without sequelae to chronic, complicated cases associated with several long-term effects. Regardless of the possible etiologic factors this extremely varying course of the disease may be considered as a result of the balance between the extent of the destructive processes associated with the inflammation and the local reconstructive capabilities and defenses. To understand these interacting and competitive forces a more profound knowledge of the inflammatory process is necessary. During the last two decades information has accumulated concerning the involvement of oxygen-derived free radicals in different pathologic processes, inflammation in particular ( 6 - 14). According to this knowledge stimulated phagocytes release oxygen-derived free radicals into the extra-cellular space with the rational purpose to eliminate micro-organisms, bacterial products, foreign material, cell debris, etc. Simultaneously, however, these highly reactive and toxic molecules also exert damage on the normal tissue cells. To counter the toxicity accompanying the radicals, a defense system is available in the tissue, partly by the release of anti-oxidative enzymes from the phagocytes, partly by the enzymes inside the normal tissue cells. Thus, basically the outcome of the inflammatory process might be a biochemical balance between the amount of oxygen-derived free radicals and the capacity of the defense mechanisms.
1018
T. Oiyesen and J. D. BorgIum
Acta Otolaryngol (Stockh) 112
Downloaded by [University of California Santa Barbara] at 18:49 05 May 2016
Hitherto the oxygen-derived free radicals have attracted only sparse attention within the otologic field. Nevertheless it seems relevant to accumulate knowledge on this subject since the most common middle ear disease. SOM, is characterized by inflammation. Instability and high reactivity make the radicals inaccessible to direct laboratory measurements. Consequently the generation of oxygen-derived free radicals can only be estimated indirectly by measuring the antioxidative enzymes of which superoxide dismutase (SOD) is the most frequently applied ( 15-20). The aim of the present study was to clarify if SOD is detectable in the middle ear fluid from children with untreated SOM and an intact tympanic membrane. MATERIAL AND METHOD Cfieniicals The following chemicals were required for the SOD measurements and were all obtained from Sigma Chemicals, St. Louis, MO, USA: luminol (5-amino-2,3-dihydro-1,4-phthalazinedione, A-851 1). hypoxanthine (&hydroxypurine, H-9377), xanthine oxidase (X-1875), bovine albumin (A-7030) and standard superoxide dismutase (3000 U/mg protein, S-7006). Equipmeni
The SOD measurements were performed with a luminescence photometer (1251 Luminometer, LKB Wallac) which detects photon emission from chemical reactions and converts the values into voltage (mV). The assay was run with the measurement mode for continuous recording and at 25’C. Each of 25 samples was measured twice for 10 s in one assay using 3 mi polysterene test cuvettes (LKB Wallac) placed in the constantly rotating cuvette container. The duration of a single rotation was 10 min. The assay was run for 120 min, i.e. each sample was measured 12 times with a delay of 10 min. Patients
Sixty-six children with SOM during at least 3 successive months were included. The diagnosis was verified by otomicroscopy and the presence of a flat curve obtained by tympanometry. The median age was 4 years, range: 1-14 years. Twenty-three were girls, 43 boys. In 17 children the disease was bilateral, in 49 it was found only unilateral. The middle ear fluid from a total of 83 ears was collected in sterile syringes connected to a middle ear suction in relation to insertion of ventilation tubes. The viscosity of the samples was mucoid in all ears except 5, which had glue ears. The samples were stored at - 70°C for the luminol assay. Postoperatively the children underwent ENT examination with otomicroscopy and, if possible, audiometry after 2 weeks and every third month for a year. Patients with a non-purulent middle ear effusion for more than 2 weeks were registered as patients with a poor outcome (referred to as “complication”). Assay The applied assay was based upon a modification of the methods described by Puget et al. ( 19) and Bensinger et al. (15,16) dealing with luminol chemiluminescence. Principally the method involves generation of superoxide anions, 0; , in a luminol containing buffer. The reaction between luminol and the anions results in emission of photons. Addition of superoxide dismutase reduces the amount of anions available for luminol and the photon emission will decline. The extent of this reduction is proportional to the concentration of superoxide dismutase.
Downloaded by [University of California Santa Barbara] at 18:49 05 May 2016
Acta Otolaryngol (Stockh) I12
Superoxide dismutase in middle ear Puid
The following solutions and reagents were prepared for the assay: 1) Luminol containing buffer: 20 mM Tris-HC1, pH: 7.8, 0.1 mM EDTA, 25 mM luminol (from a stock solution in 0.1 M NaOH) and 0.1 mg/ml bovine albumin. The buffer was incubated 24 h at 4°C equilibrating with atmospheric air. To determine the optimal pH of the reaction, 3 different pH values were chosen: 7.8, 8.7 and 1 I . One and a half ml of the luminol containing buffer in duplicate were measured in the assay; 2) To one litre of the luminol containing buffer were added hypoxanthine and xanthine oxidase to a final concentration of 1.7 mM and 0.32 U/I, respectively. Aliquots of 1.5 ml in duplicate were measured in the assay exhibiting the resulting photon emission from the non-inhibited reaction (NIR) with luminol; 3) To obtain a standard curve showing the inhibition of photon emission the standard superoxide dismutase (st. SOD) was diluted in pyogen free water and to 75 pl were added 1.5 ml luminol containing buffer with hypoxanthine and xanthine oxidase resulting in final concentrations of st. SOD of 100, 300, 750 and 1,000 ng per 1.5 ml solution. Each standard was measured in duplicate. The standard curve was prepared from the voltage values after 20 min in the assay; 4) A series of dilutions (l:l-l:lO) of the samples was prepared by pyogen free water and to 75 pl were added 1.5 ml luminol containing buffer with hypoxanthine and xanthine oxidase. The samples were measured in duplicate. The content of superoxide dismutase in the samples was extra-polated from the standard curve. To determine the reproducibility of the method, 14 samples were measured twice with several days’ interval. The samples were stored at -70°C between each assay. The results are described by the range, the median and the 95% confidence limits. The Mann-Whitney U-test and the Student’s t-test were used for analyzing the data. To estimate the variance of the duplicate measurements and the reproducibility of the method, i.e. the day to day variance, the mean and the standard error of the mean were calculated.
RESULTS The assay. Fig. 1 demonstrates the time related course of the photon emission when to the standard SOD (left) and the samples (right) are added the luminol containing buffer in which 0,-is generated. NIR symbolizes the basic reaction whereby 0, is made (non-inhibited reaction). The resulting photon (light) emission declines in relation to time in a linear way. Addition of less than 100 ng st. SOD did not result in any detectable inhibition of photon emmision. In other words, the detectability of the present method is 100 ng per 1.5 ml, i.e. 67 ng/ml. Addition of more than 100 ng st. SOD inhibited the light emission demonstrated by a parallel displacement of the NIR curve. The maximum inhibition of the photon emission in the present study was achieved by addition of 1,000 ng st. SOD. The displacement appears to be exponentially dependent upon the added SOD dose, which is shown in Fig. 2. Furthermore the assay was pH dependent: the higher the pH value, the lower rate of the chemical process and exhibiting of an optimum pH of 7.8. The standard curves in Fig. 3 are depicted based on the results shown in Fig. 1 and the concentration of SOD in the samples is read given the value of light emission at the 20 min-point. The duplicate measurements of both the standards and the samples show acceptable concordance with an average standard error of the mean (SEM)below 10%. On the other hand, the day-to-day variance was considerable (SEM: 20-30%); the standards and the samples being within the Same range. Therefore it is necessary to run a series of standards with any series of samples. The samples. As demonstrated in Fig. 4 the estimated SOD concentrations in the middle ear fluid (MEF) ranged from 0 and 12,000 ng/mI with a binomial distribution. The median SOD concentration was 340 ng/ml and 95% confidence limits were between 0 and 1,500 ng/
1019
1020 T. Oiiesen and J. D. Borglum
-
82:
:... ....-...... .. -...-.. .....-.. . ........ .-.. ..., . ..... .
........... ..
....r
------_--Downloaded by [University of California Santa Barbara] at 18:49 05 May 2016
--*
Acta Otolaryngol (Stockh) 112
-
E
5000
0 c
.Downloaded by [University of California Santa Barbara] at 18:49 05 May 2016
v)
.-E
c, L m
.-
Fig. 3. Standard curves at time= 20min. pH =7.8 from 3 different
-I
days in a logarithmic plot showing a linear relation between light emission and amount of SOD.The dotted lines indicate reading of the concentration of SOD in a sample.
I
t
1000
.
.
.
.
.
.
500
100
1000
st.SO0
(ng)
Nurnber o f HEF's
0
I
-E
\
m E
3
ISSN: 0001-6489 (Print) 1651-2251 (Online) Journal homepage: http://www.tandfonline.com/loi/ioto20
Superoxide Dismutase in Middle Ear Fluid from Children with Secretory Otitis Media Therese Ovesen & Jens D. Börglum To cite this article: Therese Ovesen & Jens D. Börglum (1992) Superoxide Dismutase in Middle Ear Fluid from Children with Secretory Otitis Media, Acta Oto-Laryngologica, 112:6, 1017-1024, DOI: 10.3109/00016489209137504 To link to this article: http://dx.doi.org/10.3109/00016489209137504
Published online: 08 Jul 2009.
Submit your article to this journal
Article views: 1
View related articles
Citing articles: 1 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ioto20 Download by: [University of California Santa Barbara]
Date: 05 May 2016, At: 18:49
Acta Otolaryngol (Stockh) 1992; 112: 1017-1024
Superoxide Dismutase in Middle Ear Fluid from Children with Secretory Otitis Media THERESE OVESEN' and JENS D. BORGLUM*
Downloaded by [University of California Santa Barbara] at 18:49 05 May 2016
From the 'ENT Department, University Hospital of Aarhus, and 'University Clinic of Endocrinology and Internal Medicine, Aarhus County Hospital, Aarhus, Denmark
Ovensen T, Borglum JD. Superoxide dismutase in middle ear fluid from children with secretory otitis media. Acta Otolaryngol (Stockh) 1992; 1 1 2 1017-1024. Superoxide dismutase (SOD) constitutes an important enzymatic defense against the destructive biochemical events associated with inflammation. To examine the presence of SOD in the middle ear fluid (MEF) in secretory otitis media (SOM) the content of SOD was measured in the MEF from 66 children with SOM. The applied method was based on light emission associated with reduction of the chemiluminescence of luminol reacting with superoxide anions (OF). From the inhibition exerted by the addition of standard SOD the concentration of SOD in the samples was extrapolated. The findings divided the MEF's into two significantly different groups: 48% with undetectable SOD, 52% with a SOD range of 200-12,000 ng/ml, median: 3,333 ng/ml, 95% confidence limits: 2,000-5,ooO ngjml ( p < 0.0005). It is suggested that the concentration of superoxide dismutase in the middle ear may play an essential role for the outcome of secretory otitis media and the effect of insertion of ventilation tubes. Key words: superoxide dismutase, secretory otitis media.
INTRODUCTION Secretory otitis media (SOM) is an inflammatory middle ear disease characterized by secretory transformation of the epithelium lining the middle ear cavity and subepithelial oedema, infiltration of phagocytes, vascular proliferation and hypertrophia/hyperplasia of the connective tissue ( 1-4). Furthermore, SOM is accompanied by fluid in the cavity, which may result from either transudation or exudation and active secretion from the epithelial cells or a combination of these processes ( 1-4). Etiologically and pathogenetically both dysfunction of the Eustachian tube resulting in a negative middle ear pressure and infectious spreading from the nasopharynx seem to be involved (3, 4, 5). The clinical picture ranges from transient, spontaneously healing episodes without sequelae to chronic, complicated cases associated with several long-term effects. Regardless of the possible etiologic factors this extremely varying course of the disease may be considered as a result of the balance between the extent of the destructive processes associated with the inflammation and the local reconstructive capabilities and defenses. To understand these interacting and competitive forces a more profound knowledge of the inflammatory process is necessary. During the last two decades information has accumulated concerning the involvement of oxygen-derived free radicals in different pathologic processes, inflammation in particular ( 6 - 14). According to this knowledge stimulated phagocytes release oxygen-derived free radicals into the extra-cellular space with the rational purpose to eliminate micro-organisms, bacterial products, foreign material, cell debris, etc. Simultaneously, however, these highly reactive and toxic molecules also exert damage on the normal tissue cells. To counter the toxicity accompanying the radicals, a defense system is available in the tissue, partly by the release of anti-oxidative enzymes from the phagocytes, partly by the enzymes inside the normal tissue cells. Thus, basically the outcome of the inflammatory process might be a biochemical balance between the amount of oxygen-derived free radicals and the capacity of the defense mechanisms.
1018
T. Oiyesen and J. D. BorgIum
Acta Otolaryngol (Stockh) 112
Downloaded by [University of California Santa Barbara] at 18:49 05 May 2016
Hitherto the oxygen-derived free radicals have attracted only sparse attention within the otologic field. Nevertheless it seems relevant to accumulate knowledge on this subject since the most common middle ear disease. SOM, is characterized by inflammation. Instability and high reactivity make the radicals inaccessible to direct laboratory measurements. Consequently the generation of oxygen-derived free radicals can only be estimated indirectly by measuring the antioxidative enzymes of which superoxide dismutase (SOD) is the most frequently applied ( 15-20). The aim of the present study was to clarify if SOD is detectable in the middle ear fluid from children with untreated SOM and an intact tympanic membrane. MATERIAL AND METHOD Cfieniicals The following chemicals were required for the SOD measurements and were all obtained from Sigma Chemicals, St. Louis, MO, USA: luminol (5-amino-2,3-dihydro-1,4-phthalazinedione, A-851 1). hypoxanthine (&hydroxypurine, H-9377), xanthine oxidase (X-1875), bovine albumin (A-7030) and standard superoxide dismutase (3000 U/mg protein, S-7006). Equipmeni
The SOD measurements were performed with a luminescence photometer (1251 Luminometer, LKB Wallac) which detects photon emission from chemical reactions and converts the values into voltage (mV). The assay was run with the measurement mode for continuous recording and at 25’C. Each of 25 samples was measured twice for 10 s in one assay using 3 mi polysterene test cuvettes (LKB Wallac) placed in the constantly rotating cuvette container. The duration of a single rotation was 10 min. The assay was run for 120 min, i.e. each sample was measured 12 times with a delay of 10 min. Patients
Sixty-six children with SOM during at least 3 successive months were included. The diagnosis was verified by otomicroscopy and the presence of a flat curve obtained by tympanometry. The median age was 4 years, range: 1-14 years. Twenty-three were girls, 43 boys. In 17 children the disease was bilateral, in 49 it was found only unilateral. The middle ear fluid from a total of 83 ears was collected in sterile syringes connected to a middle ear suction in relation to insertion of ventilation tubes. The viscosity of the samples was mucoid in all ears except 5, which had glue ears. The samples were stored at - 70°C for the luminol assay. Postoperatively the children underwent ENT examination with otomicroscopy and, if possible, audiometry after 2 weeks and every third month for a year. Patients with a non-purulent middle ear effusion for more than 2 weeks were registered as patients with a poor outcome (referred to as “complication”). Assay The applied assay was based upon a modification of the methods described by Puget et al. ( 19) and Bensinger et al. (15,16) dealing with luminol chemiluminescence. Principally the method involves generation of superoxide anions, 0; , in a luminol containing buffer. The reaction between luminol and the anions results in emission of photons. Addition of superoxide dismutase reduces the amount of anions available for luminol and the photon emission will decline. The extent of this reduction is proportional to the concentration of superoxide dismutase.
Downloaded by [University of California Santa Barbara] at 18:49 05 May 2016
Acta Otolaryngol (Stockh) I12
Superoxide dismutase in middle ear Puid
The following solutions and reagents were prepared for the assay: 1) Luminol containing buffer: 20 mM Tris-HC1, pH: 7.8, 0.1 mM EDTA, 25 mM luminol (from a stock solution in 0.1 M NaOH) and 0.1 mg/ml bovine albumin. The buffer was incubated 24 h at 4°C equilibrating with atmospheric air. To determine the optimal pH of the reaction, 3 different pH values were chosen: 7.8, 8.7 and 1 I . One and a half ml of the luminol containing buffer in duplicate were measured in the assay; 2) To one litre of the luminol containing buffer were added hypoxanthine and xanthine oxidase to a final concentration of 1.7 mM and 0.32 U/I, respectively. Aliquots of 1.5 ml in duplicate were measured in the assay exhibiting the resulting photon emission from the non-inhibited reaction (NIR) with luminol; 3) To obtain a standard curve showing the inhibition of photon emission the standard superoxide dismutase (st. SOD) was diluted in pyogen free water and to 75 pl were added 1.5 ml luminol containing buffer with hypoxanthine and xanthine oxidase resulting in final concentrations of st. SOD of 100, 300, 750 and 1,000 ng per 1.5 ml solution. Each standard was measured in duplicate. The standard curve was prepared from the voltage values after 20 min in the assay; 4) A series of dilutions (l:l-l:lO) of the samples was prepared by pyogen free water and to 75 pl were added 1.5 ml luminol containing buffer with hypoxanthine and xanthine oxidase. The samples were measured in duplicate. The content of superoxide dismutase in the samples was extra-polated from the standard curve. To determine the reproducibility of the method, 14 samples were measured twice with several days’ interval. The samples were stored at -70°C between each assay. The results are described by the range, the median and the 95% confidence limits. The Mann-Whitney U-test and the Student’s t-test were used for analyzing the data. To estimate the variance of the duplicate measurements and the reproducibility of the method, i.e. the day to day variance, the mean and the standard error of the mean were calculated.
RESULTS The assay. Fig. 1 demonstrates the time related course of the photon emission when to the standard SOD (left) and the samples (right) are added the luminol containing buffer in which 0,-is generated. NIR symbolizes the basic reaction whereby 0, is made (non-inhibited reaction). The resulting photon (light) emission declines in relation to time in a linear way. Addition of less than 100 ng st. SOD did not result in any detectable inhibition of photon emmision. In other words, the detectability of the present method is 100 ng per 1.5 ml, i.e. 67 ng/ml. Addition of more than 100 ng st. SOD inhibited the light emission demonstrated by a parallel displacement of the NIR curve. The maximum inhibition of the photon emission in the present study was achieved by addition of 1,000 ng st. SOD. The displacement appears to be exponentially dependent upon the added SOD dose, which is shown in Fig. 2. Furthermore the assay was pH dependent: the higher the pH value, the lower rate of the chemical process and exhibiting of an optimum pH of 7.8. The standard curves in Fig. 3 are depicted based on the results shown in Fig. 1 and the concentration of SOD in the samples is read given the value of light emission at the 20 min-point. The duplicate measurements of both the standards and the samples show acceptable concordance with an average standard error of the mean (SEM)below 10%. On the other hand, the day-to-day variance was considerable (SEM: 20-30%); the standards and the samples being within the Same range. Therefore it is necessary to run a series of standards with any series of samples. The samples. As demonstrated in Fig. 4 the estimated SOD concentrations in the middle ear fluid (MEF) ranged from 0 and 12,000 ng/mI with a binomial distribution. The median SOD concentration was 340 ng/ml and 95% confidence limits were between 0 and 1,500 ng/
1019
1020 T. Oiiesen and J. D. Borglum
-
82:
:... ....-...... .. -...-.. .....-.. . ........ .-.. ..., . ..... .
........... ..
....r
------_--Downloaded by [University of California Santa Barbara] at 18:49 05 May 2016
--*
Acta Otolaryngol (Stockh) 112
-
E
5000
0 c
.Downloaded by [University of California Santa Barbara] at 18:49 05 May 2016
v)
.-E
c, L m
.-
Fig. 3. Standard curves at time= 20min. pH =7.8 from 3 different
-I
days in a logarithmic plot showing a linear relation between light emission and amount of SOD.The dotted lines indicate reading of the concentration of SOD in a sample.
I
t
1000
.
.
.
.
.
.
500
100
1000
st.SO0
(ng)
Nurnber o f HEF's
0
I
-E
\
m E
3
