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Salivary oxidative stress in children during acute asthmatic attack and during remission
ARTICLE IN PRESS Respiratory Medicine (2006) 100, 1195–1201
Salivary oxidative stress in children during acute asthmatic attack and during remission Lea Bentura,b,, Yasser Mansoura, Riva Brikb,c, Yoav Eizenberga, Rafael M. Naglerb,d a
Pediatric Pulmonology Unit, Meyer Children’s Hospital, Rambam Medical Center, P.O. Box 9602, Haifa 31092, Israel b Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel c Pediatric Department, Meyer Children’s Hospital, Rambam Medical Center d Department of Oral and Maxillofacial Surgery, Oral Biochemistry Laboratory and Salivary Clinic, Rambam Medical Center Received 9 June 2005; accepted 22 October 2005
KEYWORDS Asthma; Saliva; Oxidative stress
Summary Background: Patients with asthma generate an increased amount of reactive oxygen species from peripheral blood cells, which may contribute to its pathogenesis. Saliva analysis is non-invasive and friendly to children. We undertook this study to analyze the salivary oxidative profile and composition in children with asthma during attack and remission, and to compare them with the levels of salivary antioxidants of healthy control children. Methods: School age (range 6–18 years) children referred to the emergency room for acute asthma were included. Clinical score was assessed, spirometry performed, and saliva samples were collected and analyzed. All measurements were repeated during remission of asthma attack (2–4 weeks after attack). Salivary analysis was performed blindly during asthma attack and the results were compared to those obtained during remission, and to those of the control group. Results: Statistically significant decreases in levels of salivary peroxidase enzyme activity were observed in asthmatic children during attack compared with healthy controls, with partial recovery during remission of attack. Similarly decreased levels of calcium concentrations were observed in asthmatic children, accompanied by increased phosphate levels. Conclusions: Children with acute asthma attacks exhibit a decrease in the activity of the most important salivary antioxidant enzyme-peroxidase, which is accompanied by other salivary composition alterations. Hence, acute asthma is manifested
Corresponding author. Pediatric Pulmonology Unit, Meyer Children’s Hospital, Rambam Medical Center, P.O. Box 9602, Haifa 31092,
Israel. Tel.: +972 4 8543263; fax: +972 4 8543127. E-mail address: [email protected] (L. Bentur). 0954-6111/$ - see front matter & 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.rmed.2005.10.022
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L. Bentur et al. by salivary changes. This implies systemic oxidative stress in asthma, which may be reflected in salivary analysis. & 2005 Elsevier Ltd. All rights reserved.
Introduction Asthma is a chronic inflammatory airway disease.1 The inflammatory cells infiltrating the airways produce several mediators that modulate the inflammatory response. In recent years, oxidative stress has been increasingly recognized as one of the major factors contributing to the chronic inflammatory process. Furthermore, there is an increase in generation of oxidants and lipid peroxidation products in the plasma, including a range of toxic reactive oxygen species (ROS) such as superoxide radical, hydrogen peroxide, hypochlorous acid and hydroxyl radical.2–5 Moreover, these ROS can react with nitrite and nitric oxide (NO) to form reactive nitrogen species (RNS). ROS have been shown to be associated with many pathophysiological changes that are relevant in asthma, such as increased lipid peroxidation, increased airway reactivity and secretions, increased production of chemoattractants, and increased vascular permeability.6–8 The elevated oxidative stress in asthma is also reflected by increased production of protein carbonyls in plasma,9 increased plasma isoprostanes,10 increased oxidized glutathione in bronchoalveolar lavage (BAL) fluid,11 and increased production of NO in exhaled air.12 The lungs and blood contain several antioxidants to counter oxidant-mediated toxicity, including superoxide dismutase (SOD), catalase, glutathione, glutathione peroxidase (GSH-Px), catalase and vitamins.13–16 Changes in antioxidant defenses have been reported, including decreased GSH-Px in whole blood, plasma, and platelets, a deficiency of selenium,17–19 decreased protein sulfhydrils and total antioxidant capacity in plasma,19 decreased SOD activity in BAL cells,20 and decreased vitamin C and vitamin E concentration in BAL fluid.11 Human saliva has a profound antioxidant capacity. The salivary antioxidant system includes various molecules and enzymes, of which the most important are the uric acid molecule and the peroxidase enzyme (salivary peroxidase (SPO)), both of which are water-soluble. The lipid-soluble antioxidants carried by lipoproteins, whose concentration in saliva is very low, contribute no more than 10% of the total salivary antioxidant capacity.21,22 Uric acid, the most important antioxidant molecule in saliva,22–24 contributes approximately 70% of the total salivary antioxidant capacity,
according to Moore,22 while ascorbic acid molecules play a role as secondary antioxidants. To the best of our knowledge, the levels of antioxidants in the saliva of asthmatic children have not been previously studied. The aim of this study was to analyze the antioxidant status of saliva in children with asthma during attack and remission, and to make a comparison with the levels of salivary antioxidants in healthy control children.
Patients & methods Patients Children presenting to the Pediatric Emergency Room (ER) of Meyer Children’s Hospital with acute asthma were eligible to participate in the study. Inclusion criteria were: age 6–18 years, asthma diagnosed according to GINA (Global Initiative for Asthma) 2000 guidelines, and ability to perform spirometry consistently. Exclusion criteria were: acute gingivitis, acute febrile illness or clinical pneumonia, any other chronic lung disease, and administration of anti-inflammatory agents (inhaled/ systemic corticosteroids or antileukotrienes) within 2 weeks or bronchodilator administration less than 4 h prior to enrollment. Each subject was assessed during the acute presentation to the ER, and 2–4 weeks later. Assessment consisted of a clinical score (CS), spirometry, sialometry and sialochemistry.
Clinical assessment CS included respiratory rate, retractions, oxygen saturation, inspiratory/expiratory (I/E) ratio (estimated by auscultation) and presence of inspiratory or expiratory wheezing, each graded from 0 to 3, maximum of 15 (Table 1).25 All measurements during attacks and remissions were performed in the Pediatric Pulmonology Unit, Rambam Medical Center, by the same investigator (LB) for consistency. Spirometry: forced expiratory volume in 1 s (FEV1) was measured with a Vitalograph Alpha spirometer (Vitalograph, Buckingham, UK). Healthy children without any history or symptoms of asthma served as a control group.
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Table 1
Clinical scores of asthmatic patients—each variable is graded between 0 and 3.
Score
Wheezing
Insp/Exp ratio
Oxygen saturation
Accessory muscles use
Respiratory rate
0 1 2 3
None End expiratory Throughout expiration Expiratory and inspiratory
2:1 1:1 1:2 1:3
99–100 96–98 93–95 o93
+ ++ +++
o20 20–35 36–50 450
Salivary analysis Unstimulated whole saliva samples were obtained and no oral stimulus was permitted for 60 min prior to collection, as previously described.24 Following collection on ice, the salivary samples were frozen at 20 1C for subsequent analysis. Salivary gland flow rates were expressed as volume of saliva (mL) secreted per minute. The general sialochemistry analysis was performed as previously described, including: pH, calcium (Ca), phosphate (P), magnesium (Mg), zinc (Zn), uric acid (UA), total protein (TP), albumin (Alb), lactate dehydrogenase (LDH) and amylase (Amy).24 The salivary analysis also included the following antioxidants: peroxidase, SOD, uric acid and total antioxidant status (TAS) concentration.26,27
assay, uric acid is transformed by uricase into allantoin and hydrogen peroxide that, under the catalytic influence of peroxidase, oxidizes the chromogen (4-minophenazone/N-ethyl-methylanilin propan-sulphonate sodic) to form a red compound, whose intensity of color is proportional to the amount of uric acid present in the sample, which is read at a wavelength of 546 nm.
Salivary total antioxidant status (TAS) The assay used was based on a commercial kit supplied by Randox (USA) in which metmyoglobin in the presence of iron is turned into ferrylmyoglobin. Incubation of the latter with the Randox reagent ABTS results in the formation of a blue-green colored radical which can be detected at 600 nm.24
Salivary peroxidase (SPO) activity Statistical analysis SPO activity was measured using the thionitrobenzoicacid (NBS) assay. Briefly, the calorimetric change induced by the reaction between the enzyme and the substrate, Dithiobis 2-Nitrobensoic Acid (DTNB) in the presence of mercaptoethanol, was read at 412 nm wavelength for 20 s.24
Salivary superoxide dismutase (SOD) activity Total activity of SOD isoenzymes (Cu0 -{1-[(phenylamino)-carbonyl]-3, 4-tetrazolium}-bis (4-methoxy6-nitro) benzenesulfonic acid hydrate reduction by xanthine–xanthine oxidase. The method is a modification of the NBT assay. Xtt is reduced by the superoxide anion (O 2 .) that is generated by xanthine oxidase. The formazan is read at 470 nm. SOD inhibits this reaction by scavenging the .O2. One unit of the enzyme is defined as the amount of enzyme needed for 50% inhibition of absorption in the absence of the enzyme.27
Salivary uric acid concentration Uric acid concentration was measured with a kit supplied by Sentinel CH (Milan, Italy).24 In this
Data concerning the levels of various parameters evaluated in whole saliva are expressed as means and ranges, and median7SEM. Paired t-test was used to compare salivary composition and antioxidant levels between asthma attack and remission. Unpaired t-test was used to compare between patients and control groups. Spearman’s rank coefficient of correlation was used for analyzing saliva and the various clinical and spirometric parameters. Statistical significance was set at Po0:05.
Results Twelve asthmatic children were recruited to the study, and were compared to 14 control patients. All asthmatic children presented to the ER with an acute exacerbation of asthma. Their ages ranged from 6 to 18 years, with a mean7SD age of 9.7573.22 years. Nine patients had clinical manifestations of atopy: allergic rhinitis (9/9), atopic dermatitis (4/9), and allergic conjunctivitis (6/9). Seven patients had a family history of asthma and seven had passive smoking exposure.
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L. Bentur et al. Clinical scores and FEV1 during exacerbation and remission of asthma.
N ¼ 12
Mean7SD
(Range)
Age (years) CS, attack CS, remission (N ¼ 10) FEV1, attack (% predicted) FEV1, remission (% predicted) (N ¼ 10)
9.7573.22 6.9274.58 1.570.71 61719.01 92.3722.33
6–16 1–14 1–3 34–87 66–139
CS—clinical score; FEV1—forced expiratory volume in 1 s. Po0:05.
All children received an oral tapering course of methylprednisolone (initial dose of 1–2 mg/kg) followed by maintenance therapy with budesonide Turbohaler (200 mcg twice daily). CSs decreased from 6.9274.58 during attack to 1.570.71 (mean7SD) on remission (Po0:0001), while FEV1 rose from 61719.01 to 92.3722.33 percent of predicted, respectively (Po0:0001) (Table 2).
Sialometry and sialochemistry The mean salivary flow rate value and pH of the entire group of patients was within normal values and did not differ from the healthy controls. The sialochemical analysis demonstrated a significant decrease of calcium and increase of phosphate concentrations in the asthmatic children (Tables 3 and 4). The amylase activity was twofold higher in asthmatic children during attack (P ¼ 0:09). No significant differences were found between patients and controls regarding total protein, albumin levels, and LDH activity (Tables 3 and 4).
Salivary antioxidant analysis The salivary antioxidant analysis revealed a significant decrease in the levels of SPO in asthmatic patients during acute asthma attack compared with controls (Fig. 1). The levels of SPO increased during remission of attack without reaching control levels, although this difference was statistically insignificant. A similar trend toward increasing levels while in remission was observed for all other antioxidants (TAS, uric acid and SOD). However, it also did not achieve statistical significance. Thus, the SOD activity values and TAS levels dropped by 26% and 28%, respectively, in asthmatic patients during attack, as compared to healthy controls. Following remission, these values fully recovered (Table 3). No significant differences were found between patients and controls regarding salivary uric acid,
yet the uric acid concentration following remission increased by 39% (Table 4). No correlation was found between saliva antioxidant levels and FEV1 or CS values.
Discussion The main finding of this study is that SPO was shown to be reduced during acute asthmatic attack. This reduction may result from an increase in the level of oxidants, which reacted with the enzyme.28,29 This may be of paramount importance, since SPO is considered the major salivary antioxidant enzyme. All other antioxidants measured exhibited a similar trend, with increased levels during remission, although not of statistical significance. The mechanism by which the decreased levels of calcium concentration and increased phosphate levels occurred in asthmatic children remains obscure and should be further elucidated. The decreased levels of SPO and calcium, and increased level of phosphate observed are in accord with the studies published by Ryberg et al.30 who reported changes in the salivary composition of asthmatic patients treated with beta 2-adrenoceptor agonists. This significant decrease in the SPO leaves the oral cavity exposed to oxidative stress. Moreover, Hyyppa et al.31 reported that asthmatic children had more gingivitis than their healthy controls. Previous studies32–34 suggested an association between salivary antioxidant activity and periodontal disease. Asthma, as well as other pulmonary diseases, have long been associated with inflammation, and recently with oxidative stress. Oxidative stress is the final result of numerous molecular pathways involving ROS and RNS, which may lead to the observed increased airway reactivity and secretions, increased production of chemoattractants, and increased vascular permeability.6–8 Although the aforementioned oxidative process mainly affects
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Table 3 Comparison of salivary pH, electrolytes, protein, enzyme levels and antioxidant activity levels between healthy controls and asthmatic patients during remission.
pH Ca (mg/dL) P (mg/dL) Mg (mg/dL) Zn (mg/dL) TP (mg/dL) ALB (mg/dL) LDH(U/L) AMY (IU/L) Uric acid (mg/dL) SPO (U/100 mL) TAS (mmol/L) SOD (U/mL)
Median (range) Mean7SEM Median (range) Mean7SEM Median (range) Mean7SEM Median (range) Mean7SEM Median (range) Mean7SEM Median (range) Mean7SEM Median (range) Mean7SEM Median (range) Mean7SEM Median (range) Mean7SEM Median (range) Mean7SEM Median (range) Mean7SEM Median (range) Mean7SEM Median (range) Mean7SEM
Healthy (n ¼ 14)
Asthma in remission (n ¼ 12)
7.00 (6.4–7.3) 6.9470.05 2.35(0.5–59.7) 6.6974.10 11.4 (4.9–1.5) 14.2473.00 0.95 (0.4–1.9) 1.0070.13 132 (21–586) 165741.60 67.05 (16.6–100.8) 62.2076.23 39.4 (10.1–247.4) 61.65716.25 422 (42–912) 424778 663 (110–1192) 716780.8 1.83 (0.32–20.38) 3.3871.35 580 (420–890) 600740 0.5 (0.13–1.06) 0.570.07 1.71 (0.32–7.34) 2.1570.55
7.00 (6.4–7.0) 6.8770.07 0.70 (0.2–1.2) 0.7470.11 14.2 (9.5–20.2) 14.4071.07 0.7 (0.2–1.2) 0.7270.11 145 (7.0–314) 144734.50 71.1(44.4–126.6) 7477.90 51.3 (7.2–83.9) 47.9077.87 509 (108–831) 509776.00 830 (179.3–2802) 9907406. 2.47 (0.62–3.66) 2.1870.36 540 (430–670) 540748 0.61 (0.24–0.95) 0.6070.09 1.71 (0.34–4.66) 1.9270.62
Results are expressed as medians and range, means7SEM (standard error). Po0:05.
the respiratory system, changes in the oxidative– antioxidative balance can also be seen in the plasma and blood cells.3,9,10. With the profound antioxidative capacity of human saliva in mind, our study aimed to evaluate salivary oxidative status in asthmatic children. The current study demonstrates that asthmatic children have decreased salivary antioxidant levels during acute asthma exacerbations, compared to healthy children, as reflected by decreased salivary SPO levels. Similar to our finding, Smith et al.20 found decreased antioxidant level (SOD) activity in BAL cells of asthmatic patients, while two recently published works found increased SOD activity in asthmatic patients’ erythrocytes.28,29 The difference in the findings may be explained by the difference in the tissues obtained. Recently, Schock et al.35 demonstrated significantly lower levels of ascorbic acid in induced sputum and saliva compared with plasma, and suggested that there is no free diffusion of ascorbic acid between the vascular-interstitial fluid compartments and the tracheobronchial airway secretions.
Reviewing antioxidant function in the oxidative process shows that it has conflicting functions: on the one hand, it participates in the scavenging of the potent superoxide radical, but on the other hand, it creates the hydrogen peroxide radical which propagates further ROS and RNS creation. The net result of these conflicting functions is naturally determined by other factors, which are not well understood and need further research for clarification. We demonstrated that SPO levels increased toward normal levels by 2–4 weeks of ambulatory anti-inflammatory therapy, in association with significant clinical and laboratory-confirmed improvement. Corticosteroids used in asthma treatment suppress airway inflammation. It is possible that anti-inflammatory therapy alleviates the oxidative load by inhibiting airway inflammation, and thereby suppresses the salivary oxidative process. This study has several limitations. The study population is relatively small. The antioxidative parameters were measured in the saliva without comparable antioxidative parameters in other body
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Table 4 Comparison of salivary pH, electrolytes, protein, enzyme levels and antioxidant activity between asthmatic patients during attack and remission.
pH Ca (mg/dL) P (mg/dL) Mg (mg/dL) Zn (mg/dL) TP (mg/dL) ALB (mg/dL) LDH (U/L) AMY (IU/L) Uric acid (mg/dL) SPO (U/100 mL) TAS (mmol/L) SOD (U/mL)
Median (range) Mean7SEM Median (range) Mean7SEM Median (range) Mean7SEM Median (range) Mean7SEM Median (range) Mean7SEM Median (range) Mean7SEM Median (range) Mean7SEM Median (range) Mean7SEM Median (range) Mean7SEM Median(range) Mean7SEM Median (range) Mean7SEM Median (range) Mean7SEM Median (range) Mean7SEM
Asthma attack (n ¼ 12)
Asthma in remission (n ¼ 12)
7.00 (6.7–7.6) 7.0070.07 0.70 (0.3–39.2) 4.3473.18 13.85 (7.5–22.8) 14.3271.10 0.65 (0.2–2.8) 0.8270.21 134 (12–331) 165741.60 66.35 (9.7–243.3) 62.2076.23 43.55 (7.6–78.8) 62.07721.6 686 (31–983) 585797 1234 (235–2674) 14367957 1.78 (0.69–2.68) 1.7370.16 500 (280–680) 480730 0.36 (0.22–1.85) 0.670.15 1.27 (0.31–7.09) 2.1570.65
7.00 (6.4–7.0) 6.8770.07 0.70 (0.2–1.2) 0.7470.11 14.2 (9.5–20.2) 14.4071.07 0.7 (0.2–1.2) 0.7270.11 145 (7.0–314) 144734.50 71.1(44.4–126.6) 7477.90 51.3 (7.2–83.9) 47.9077.87 509 (108–831) 509776.00 830 (179.3–2802) 9907406. 2.47 (0.62–3.66) 2.1870.36 540 (430–670) 540748 0.61 (0.24–0.95) 0.6070.09 1.71(0.34–4.66) 1.9270.62
Results are expressed as medians and range, means7SEM (standard error).
1000
480±30*
540±48
600±40*
SPO (U/100µL)
800 600 400 200 *p<0.05
convincing evidence that free radical mechanisms are involved in the pathogenesis of asthma and that antioxidants may be protective. Further studies involving larger groups comparing saliva, serum and BAL parameters should be conducted in order to fully understand the role of salivary antioxidants in children with asthma.
Asthma Attack Asthma Remission Healthy
0
Figure. 1 Comparison of SPO levels between healthy controls and asthmatic patients during attack and remission.
fluids such as induced sputum, BAL and blood. Furthermore, no specific inflammatory parameters were measured in addition to the clinical and spirometry evaluations. This pilot study shows that asthmatic patients have decreased levels of antioxidant enzyme activity in saliva as a result of the ongoing inflammatory and oxidative process. There is fairly
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ARTICLE IN PRESS Salivary oxidative stress in asthma 5. Vachier I, Damon M, Le Doucen C, de Paulet AC, Chanez P, Michel FB, et al. Increased oxygen species generation in blood monocytes of asthma patients. Am Rev Respir Dis 1992;146:1161–6. 6. Katsumata U, Miura M, Ichinose M, et al. Oxygen radicals produce airway constriction and hyperresponsivness in anesthetized dogs. Am Rev Respir Dis 1990;141:1158–61. 7. Abe MK, Chao TS, Solway J, Rosner MR, Hershenson MB. Hydrogen peroxide stimulates mitogen-activated protein kinase in bovine tracheal myocytes: implications for human airway disease. Am J Respir Cell Mol Biol 1994;11:577–85. 8. Wright DT, Fischer BM, Li C, Rochelle LG, Akley NJ, Adler KB. Oxidant stress stimulates mucin secretions and PLC in airway epithelium via a nitric oxide-dependent mechanism. Am J Physiol 1996;271:L854–61. 9. Rahman I, Morrison D, Donaldson K, MacNee W. Systemic oxidative stress in asthma, COPD, and smokers. Am J Respir Crit Care Med 1996;159:1055–60. 10. Wood LJ, Fitzgerald DA, Gibson PG, Cooper DM, Garg ML. Lipid peroxidation as determined by plasma isoprostanes is related to disease severity in mild asthma. Lipids 2000;35:967–74. 11. Kelly FJ, Mudway I, Blomberg A, Frew A, Sandstorm T. Altered lung antioxidant status in patients with mild asthma. Lancet 1999;354:482–3. 12. Kharitinov SA, Yates D, Robbins RA, Logan-Sinclair R, Shinebourne EA, Barnes PJ. Increased nitric oxide in exhaled air of asthmatic patients. Lancet 1994;342:133–58. 13. Agar NS, Sadrzadeh SM, Hallaway PE, Eaton JW. Erythrocyte catalase. A somatic oxidant defense? J Clin Invest 1986;77: 319–21. 14. Heffner JE, Repine JE. Antioxidants and the lung. In: Crystal RG, West WB, editors. The lung: scientific foundations. New York: Raven Press; 1991. p. 1811–20. 15. van Asbeck BS, Hoidal J, Vercellotti GM, Schwartz BA, Moldow CF, Jacob HS. Protection against lethal hyperoxia by tracheal insufflation of erythrocytes: role of red cell glutathione. Science 1985;227:756–9. 16. Bowler RP, Crapo JD. Oxidative stress in allergic respiratory diseases. J Allergy Clin Immunol 2002;11:349–56. 17. Hesselmark OL, Malmgren R, Unge G, Zetterstorm O. Lowered platelet GSH-Px activity in patients with intrinsic asthma. Allergy 1990;45:523–7. 18. Pearson DJ, Suarez-Mendez VJ, Day JP, Miller PF. Selenium status in relation to reduced glutathione peroxidase activity in aspirin sensitive asthma. Clin Exp Allergy 1991;56:43–9. 19. Picado C, Deulofen R, Lieonart R, Agusti M, Mullol J, Quinto L, et al. Dietary micronutrients/antioxidants and their relationship with bronchial asthma severity. Allergy 2001; 56:43–9. 20. Smith LJ, Shamsuddin M, Sporn PH, Denenberg M, Anderson J. Reduced superoxide dismutase in lung cells of patients with asthma. Free Radical Biol Med 1997;22:1301–7.
1201 21. Meucci E, Littarru C, Deli G, et al. Antioxidant status and dialysis: plasma and saliva antioxidant activity in patients with fluctuating urate levels. Free Radical Res 1998;29: 367–76. 22. Moore S, Calder KAC, Miller NJ, Rice-Evans CA. Antioxidant activity of saliva and periodontal disease. Free Radical Res 1994;21:417–25. 23. Kondakova I, Lissi FA, Pizarro M. Total reactive antioxidant potential in human saliva of smokers and non-smokers. Biochem Mol Biol Int 1999;6:911–20. 24. Nagler RM, Salameh F, Reznick AZ, Livshits V, Nahir AM. Salivary glands involvement in rheumatoid arthritis and its relation to the induced oxidative stress. Rheumatology 2003;42:1–8. 25. Volovitz B, Bentur L, Finkelstein Y, Mansour Y, Shalitin S, Nussinovitch M, et al. Effectiveness and safety of inhaled budesonide in controlling acute asthma attacks in children—a controlled study compared to oral prednisolone. Ann Allergy Clin Immunol 1998;102:605–60. 26. Terao J, Nagao A. Antioxidative effect of human saliva on lipid peroxidation. Agric Biol Chem 1991;55:869–72. 27. Pruitt KM, Kamau DN, Miller K, et al. Quantitative, standardized assays for determining the concentration of bovine lactoperoxidase, human salivary peroxidase, and human myeloperoxidase. Anal Biochem 1990;191:278–86. 28. Mak JC, Leung HC, Ho SP, Law BK, Lam WK, Tsang KW, et al. Systemic oxidative and antioxidative status in Chinese patients with asthma. J Allergy Clin Immunol 2004;114:260–4. 29. Hanta I, Kuleci S, Canacankatan N, Kocabas A. The oxidant–antioxidant balance in mild asthmatic patients. Lung 2003;18:347–52. 30. Ryberg M, Moller C, Ericson T. Saliva composition and caries development in asthmatic patients treated with beta 2adrenoceptor agonists: a 4-year follow-up study. Scand J Dent Res 1991;99:212–8. 31. Hyyppa TM, Koivikko A, Paunio KU. Studies on periodontal conditions in asthmatic children. Acta Odontol Scand 1979;37:15–20. 32. Moore S, Calder KA, Miller NJ, Rice-Evans CA. Antioxidant activity of saliva and periodontal disease. Free Radical Res 1994;21:417–25. 33. Sculley DV, Langley-Evans SC. Periodontal disease is associated with lower antioxidant capacity in whole saliva and evidence of increased protein oxidation. Clin Sci (Lond) 2003;105:167–72. 34. Takane M, Sugano N, Iwasaki H, Iwano Y, Shimizu N, Ito K. New biomarker evidence of oxidative DNA damage in whole saliva from clinically healthy and periodontally diseased individuals. J Periodontol 2002;73:551–4. 35. Schock BC, Koostra J, Kwack S, Hackman RM, Van Der Vliet A, Cross CE. Ascorbic acid in nasal and tracheobronchial airway lining fluids. Free Radical Biol Med 2004;37:1393–401.
Salivary oxidative stress in children during acute asthmatic attack and during remission Lea Bentura,b,, Yasser Mansoura, Riva Brikb,c, Yoav Eizenberga, Rafael M. Naglerb,d a
Pediatric Pulmonology Unit, Meyer Children’s Hospital, Rambam Medical Center, P.O. Box 9602, Haifa 31092, Israel b Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel c Pediatric Department, Meyer Children’s Hospital, Rambam Medical Center d Department of Oral and Maxillofacial Surgery, Oral Biochemistry Laboratory and Salivary Clinic, Rambam Medical Center Received 9 June 2005; accepted 22 October 2005
KEYWORDS Asthma; Saliva; Oxidative stress
Summary Background: Patients with asthma generate an increased amount of reactive oxygen species from peripheral blood cells, which may contribute to its pathogenesis. Saliva analysis is non-invasive and friendly to children. We undertook this study to analyze the salivary oxidative profile and composition in children with asthma during attack and remission, and to compare them with the levels of salivary antioxidants of healthy control children. Methods: School age (range 6–18 years) children referred to the emergency room for acute asthma were included. Clinical score was assessed, spirometry performed, and saliva samples were collected and analyzed. All measurements were repeated during remission of asthma attack (2–4 weeks after attack). Salivary analysis was performed blindly during asthma attack and the results were compared to those obtained during remission, and to those of the control group. Results: Statistically significant decreases in levels of salivary peroxidase enzyme activity were observed in asthmatic children during attack compared with healthy controls, with partial recovery during remission of attack. Similarly decreased levels of calcium concentrations were observed in asthmatic children, accompanied by increased phosphate levels. Conclusions: Children with acute asthma attacks exhibit a decrease in the activity of the most important salivary antioxidant enzyme-peroxidase, which is accompanied by other salivary composition alterations. Hence, acute asthma is manifested
Corresponding author. Pediatric Pulmonology Unit, Meyer Children’s Hospital, Rambam Medical Center, P.O. Box 9602, Haifa 31092,
Israel. Tel.: +972 4 8543263; fax: +972 4 8543127. E-mail address: [email protected] (L. Bentur). 0954-6111/$ - see front matter & 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.rmed.2005.10.022
ARTICLE IN PRESS 1196
L. Bentur et al. by salivary changes. This implies systemic oxidative stress in asthma, which may be reflected in salivary analysis. & 2005 Elsevier Ltd. All rights reserved.
Introduction Asthma is a chronic inflammatory airway disease.1 The inflammatory cells infiltrating the airways produce several mediators that modulate the inflammatory response. In recent years, oxidative stress has been increasingly recognized as one of the major factors contributing to the chronic inflammatory process. Furthermore, there is an increase in generation of oxidants and lipid peroxidation products in the plasma, including a range of toxic reactive oxygen species (ROS) such as superoxide radical, hydrogen peroxide, hypochlorous acid and hydroxyl radical.2–5 Moreover, these ROS can react with nitrite and nitric oxide (NO) to form reactive nitrogen species (RNS). ROS have been shown to be associated with many pathophysiological changes that are relevant in asthma, such as increased lipid peroxidation, increased airway reactivity and secretions, increased production of chemoattractants, and increased vascular permeability.6–8 The elevated oxidative stress in asthma is also reflected by increased production of protein carbonyls in plasma,9 increased plasma isoprostanes,10 increased oxidized glutathione in bronchoalveolar lavage (BAL) fluid,11 and increased production of NO in exhaled air.12 The lungs and blood contain several antioxidants to counter oxidant-mediated toxicity, including superoxide dismutase (SOD), catalase, glutathione, glutathione peroxidase (GSH-Px), catalase and vitamins.13–16 Changes in antioxidant defenses have been reported, including decreased GSH-Px in whole blood, plasma, and platelets, a deficiency of selenium,17–19 decreased protein sulfhydrils and total antioxidant capacity in plasma,19 decreased SOD activity in BAL cells,20 and decreased vitamin C and vitamin E concentration in BAL fluid.11 Human saliva has a profound antioxidant capacity. The salivary antioxidant system includes various molecules and enzymes, of which the most important are the uric acid molecule and the peroxidase enzyme (salivary peroxidase (SPO)), both of which are water-soluble. The lipid-soluble antioxidants carried by lipoproteins, whose concentration in saliva is very low, contribute no more than 10% of the total salivary antioxidant capacity.21,22 Uric acid, the most important antioxidant molecule in saliva,22–24 contributes approximately 70% of the total salivary antioxidant capacity,
according to Moore,22 while ascorbic acid molecules play a role as secondary antioxidants. To the best of our knowledge, the levels of antioxidants in the saliva of asthmatic children have not been previously studied. The aim of this study was to analyze the antioxidant status of saliva in children with asthma during attack and remission, and to make a comparison with the levels of salivary antioxidants in healthy control children.
Patients & methods Patients Children presenting to the Pediatric Emergency Room (ER) of Meyer Children’s Hospital with acute asthma were eligible to participate in the study. Inclusion criteria were: age 6–18 years, asthma diagnosed according to GINA (Global Initiative for Asthma) 2000 guidelines, and ability to perform spirometry consistently. Exclusion criteria were: acute gingivitis, acute febrile illness or clinical pneumonia, any other chronic lung disease, and administration of anti-inflammatory agents (inhaled/ systemic corticosteroids or antileukotrienes) within 2 weeks or bronchodilator administration less than 4 h prior to enrollment. Each subject was assessed during the acute presentation to the ER, and 2–4 weeks later. Assessment consisted of a clinical score (CS), spirometry, sialometry and sialochemistry.
Clinical assessment CS included respiratory rate, retractions, oxygen saturation, inspiratory/expiratory (I/E) ratio (estimated by auscultation) and presence of inspiratory or expiratory wheezing, each graded from 0 to 3, maximum of 15 (Table 1).25 All measurements during attacks and remissions were performed in the Pediatric Pulmonology Unit, Rambam Medical Center, by the same investigator (LB) for consistency. Spirometry: forced expiratory volume in 1 s (FEV1) was measured with a Vitalograph Alpha spirometer (Vitalograph, Buckingham, UK). Healthy children without any history or symptoms of asthma served as a control group.
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Table 1
Clinical scores of asthmatic patients—each variable is graded between 0 and 3.
Score
Wheezing
Insp/Exp ratio
Oxygen saturation
Accessory muscles use
Respiratory rate
0 1 2 3
None End expiratory Throughout expiration Expiratory and inspiratory
2:1 1:1 1:2 1:3
99–100 96–98 93–95 o93
+ ++ +++
o20 20–35 36–50 450
Salivary analysis Unstimulated whole saliva samples were obtained and no oral stimulus was permitted for 60 min prior to collection, as previously described.24 Following collection on ice, the salivary samples were frozen at 20 1C for subsequent analysis. Salivary gland flow rates were expressed as volume of saliva (mL) secreted per minute. The general sialochemistry analysis was performed as previously described, including: pH, calcium (Ca), phosphate (P), magnesium (Mg), zinc (Zn), uric acid (UA), total protein (TP), albumin (Alb), lactate dehydrogenase (LDH) and amylase (Amy).24 The salivary analysis also included the following antioxidants: peroxidase, SOD, uric acid and total antioxidant status (TAS) concentration.26,27
assay, uric acid is transformed by uricase into allantoin and hydrogen peroxide that, under the catalytic influence of peroxidase, oxidizes the chromogen (4-minophenazone/N-ethyl-methylanilin propan-sulphonate sodic) to form a red compound, whose intensity of color is proportional to the amount of uric acid present in the sample, which is read at a wavelength of 546 nm.
Salivary total antioxidant status (TAS) The assay used was based on a commercial kit supplied by Randox (USA) in which metmyoglobin in the presence of iron is turned into ferrylmyoglobin. Incubation of the latter with the Randox reagent ABTS results in the formation of a blue-green colored radical which can be detected at 600 nm.24
Salivary peroxidase (SPO) activity Statistical analysis SPO activity was measured using the thionitrobenzoicacid (NBS) assay. Briefly, the calorimetric change induced by the reaction between the enzyme and the substrate, Dithiobis 2-Nitrobensoic Acid (DTNB) in the presence of mercaptoethanol, was read at 412 nm wavelength for 20 s.24
Salivary superoxide dismutase (SOD) activity Total activity of SOD isoenzymes (Cu0 -{1-[(phenylamino)-carbonyl]-3, 4-tetrazolium}-bis (4-methoxy6-nitro) benzenesulfonic acid hydrate reduction by xanthine–xanthine oxidase. The method is a modification of the NBT assay. Xtt is reduced by the superoxide anion (O 2 .) that is generated by xanthine oxidase. The formazan is read at 470 nm. SOD inhibits this reaction by scavenging the .O2. One unit of the enzyme is defined as the amount of enzyme needed for 50% inhibition of absorption in the absence of the enzyme.27
Salivary uric acid concentration Uric acid concentration was measured with a kit supplied by Sentinel CH (Milan, Italy).24 In this
Data concerning the levels of various parameters evaluated in whole saliva are expressed as means and ranges, and median7SEM. Paired t-test was used to compare salivary composition and antioxidant levels between asthma attack and remission. Unpaired t-test was used to compare between patients and control groups. Spearman’s rank coefficient of correlation was used for analyzing saliva and the various clinical and spirometric parameters. Statistical significance was set at Po0:05.
Results Twelve asthmatic children were recruited to the study, and were compared to 14 control patients. All asthmatic children presented to the ER with an acute exacerbation of asthma. Their ages ranged from 6 to 18 years, with a mean7SD age of 9.7573.22 years. Nine patients had clinical manifestations of atopy: allergic rhinitis (9/9), atopic dermatitis (4/9), and allergic conjunctivitis (6/9). Seven patients had a family history of asthma and seven had passive smoking exposure.
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L. Bentur et al. Clinical scores and FEV1 during exacerbation and remission of asthma.
N ¼ 12
Mean7SD
(Range)
Age (years) CS, attack CS, remission (N ¼ 10) FEV1, attack (% predicted) FEV1, remission (% predicted) (N ¼ 10)
9.7573.22 6.9274.58 1.570.71 61719.01 92.3722.33
6–16 1–14 1–3 34–87 66–139
CS—clinical score; FEV1—forced expiratory volume in 1 s. Po0:05.
All children received an oral tapering course of methylprednisolone (initial dose of 1–2 mg/kg) followed by maintenance therapy with budesonide Turbohaler (200 mcg twice daily). CSs decreased from 6.9274.58 during attack to 1.570.71 (mean7SD) on remission (Po0:0001), while FEV1 rose from 61719.01 to 92.3722.33 percent of predicted, respectively (Po0:0001) (Table 2).
Sialometry and sialochemistry The mean salivary flow rate value and pH of the entire group of patients was within normal values and did not differ from the healthy controls. The sialochemical analysis demonstrated a significant decrease of calcium and increase of phosphate concentrations in the asthmatic children (Tables 3 and 4). The amylase activity was twofold higher in asthmatic children during attack (P ¼ 0:09). No significant differences were found between patients and controls regarding total protein, albumin levels, and LDH activity (Tables 3 and 4).
Salivary antioxidant analysis The salivary antioxidant analysis revealed a significant decrease in the levels of SPO in asthmatic patients during acute asthma attack compared with controls (Fig. 1). The levels of SPO increased during remission of attack without reaching control levels, although this difference was statistically insignificant. A similar trend toward increasing levels while in remission was observed for all other antioxidants (TAS, uric acid and SOD). However, it also did not achieve statistical significance. Thus, the SOD activity values and TAS levels dropped by 26% and 28%, respectively, in asthmatic patients during attack, as compared to healthy controls. Following remission, these values fully recovered (Table 3). No significant differences were found between patients and controls regarding salivary uric acid,
yet the uric acid concentration following remission increased by 39% (Table 4). No correlation was found between saliva antioxidant levels and FEV1 or CS values.
Discussion The main finding of this study is that SPO was shown to be reduced during acute asthmatic attack. This reduction may result from an increase in the level of oxidants, which reacted with the enzyme.28,29 This may be of paramount importance, since SPO is considered the major salivary antioxidant enzyme. All other antioxidants measured exhibited a similar trend, with increased levels during remission, although not of statistical significance. The mechanism by which the decreased levels of calcium concentration and increased phosphate levels occurred in asthmatic children remains obscure and should be further elucidated. The decreased levels of SPO and calcium, and increased level of phosphate observed are in accord with the studies published by Ryberg et al.30 who reported changes in the salivary composition of asthmatic patients treated with beta 2-adrenoceptor agonists. This significant decrease in the SPO leaves the oral cavity exposed to oxidative stress. Moreover, Hyyppa et al.31 reported that asthmatic children had more gingivitis than their healthy controls. Previous studies32–34 suggested an association between salivary antioxidant activity and periodontal disease. Asthma, as well as other pulmonary diseases, have long been associated with inflammation, and recently with oxidative stress. Oxidative stress is the final result of numerous molecular pathways involving ROS and RNS, which may lead to the observed increased airway reactivity and secretions, increased production of chemoattractants, and increased vascular permeability.6–8 Although the aforementioned oxidative process mainly affects
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Table 3 Comparison of salivary pH, electrolytes, protein, enzyme levels and antioxidant activity levels between healthy controls and asthmatic patients during remission.
pH Ca (mg/dL) P (mg/dL) Mg (mg/dL) Zn (mg/dL) TP (mg/dL) ALB (mg/dL) LDH(U/L) AMY (IU/L) Uric acid (mg/dL) SPO (U/100 mL) TAS (mmol/L) SOD (U/mL)
Median (range) Mean7SEM Median (range) Mean7SEM Median (range) Mean7SEM Median (range) Mean7SEM Median (range) Mean7SEM Median (range) Mean7SEM Median (range) Mean7SEM Median (range) Mean7SEM Median (range) Mean7SEM Median (range) Mean7SEM Median (range) Mean7SEM Median (range) Mean7SEM Median (range) Mean7SEM
Healthy (n ¼ 14)
Asthma in remission (n ¼ 12)
7.00 (6.4–7.3) 6.9470.05 2.35(0.5–59.7) 6.6974.10 11.4 (4.9–1.5) 14.2473.00 0.95 (0.4–1.9) 1.0070.13 132 (21–586) 165741.60 67.05 (16.6–100.8) 62.2076.23 39.4 (10.1–247.4) 61.65716.25 422 (42–912) 424778 663 (110–1192) 716780.8 1.83 (0.32–20.38) 3.3871.35 580 (420–890) 600740 0.5 (0.13–1.06) 0.570.07 1.71 (0.32–7.34) 2.1570.55
7.00 (6.4–7.0) 6.8770.07 0.70 (0.2–1.2) 0.7470.11 14.2 (9.5–20.2) 14.4071.07 0.7 (0.2–1.2) 0.7270.11 145 (7.0–314) 144734.50 71.1(44.4–126.6) 7477.90 51.3 (7.2–83.9) 47.9077.87 509 (108–831) 509776.00 830 (179.3–2802) 9907406. 2.47 (0.62–3.66) 2.1870.36 540 (430–670) 540748 0.61 (0.24–0.95) 0.6070.09 1.71 (0.34–4.66) 1.9270.62
Results are expressed as medians and range, means7SEM (standard error). Po0:05.
the respiratory system, changes in the oxidative– antioxidative balance can also be seen in the plasma and blood cells.3,9,10. With the profound antioxidative capacity of human saliva in mind, our study aimed to evaluate salivary oxidative status in asthmatic children. The current study demonstrates that asthmatic children have decreased salivary antioxidant levels during acute asthma exacerbations, compared to healthy children, as reflected by decreased salivary SPO levels. Similar to our finding, Smith et al.20 found decreased antioxidant level (SOD) activity in BAL cells of asthmatic patients, while two recently published works found increased SOD activity in asthmatic patients’ erythrocytes.28,29 The difference in the findings may be explained by the difference in the tissues obtained. Recently, Schock et al.35 demonstrated significantly lower levels of ascorbic acid in induced sputum and saliva compared with plasma, and suggested that there is no free diffusion of ascorbic acid between the vascular-interstitial fluid compartments and the tracheobronchial airway secretions.
Reviewing antioxidant function in the oxidative process shows that it has conflicting functions: on the one hand, it participates in the scavenging of the potent superoxide radical, but on the other hand, it creates the hydrogen peroxide radical which propagates further ROS and RNS creation. The net result of these conflicting functions is naturally determined by other factors, which are not well understood and need further research for clarification. We demonstrated that SPO levels increased toward normal levels by 2–4 weeks of ambulatory anti-inflammatory therapy, in association with significant clinical and laboratory-confirmed improvement. Corticosteroids used in asthma treatment suppress airway inflammation. It is possible that anti-inflammatory therapy alleviates the oxidative load by inhibiting airway inflammation, and thereby suppresses the salivary oxidative process. This study has several limitations. The study population is relatively small. The antioxidative parameters were measured in the saliva without comparable antioxidative parameters in other body
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Table 4 Comparison of salivary pH, electrolytes, protein, enzyme levels and antioxidant activity between asthmatic patients during attack and remission.
pH Ca (mg/dL) P (mg/dL) Mg (mg/dL) Zn (mg/dL) TP (mg/dL) ALB (mg/dL) LDH (U/L) AMY (IU/L) Uric acid (mg/dL) SPO (U/100 mL) TAS (mmol/L) SOD (U/mL)
Median (range) Mean7SEM Median (range) Mean7SEM Median (range) Mean7SEM Median (range) Mean7SEM Median (range) Mean7SEM Median (range) Mean7SEM Median (range) Mean7SEM Median (range) Mean7SEM Median (range) Mean7SEM Median(range) Mean7SEM Median (range) Mean7SEM Median (range) Mean7SEM Median (range) Mean7SEM
Asthma attack (n ¼ 12)
Asthma in remission (n ¼ 12)
7.00 (6.7–7.6) 7.0070.07 0.70 (0.3–39.2) 4.3473.18 13.85 (7.5–22.8) 14.3271.10 0.65 (0.2–2.8) 0.8270.21 134 (12–331) 165741.60 66.35 (9.7–243.3) 62.2076.23 43.55 (7.6–78.8) 62.07721.6 686 (31–983) 585797 1234 (235–2674) 14367957 1.78 (0.69–2.68) 1.7370.16 500 (280–680) 480730 0.36 (0.22–1.85) 0.670.15 1.27 (0.31–7.09) 2.1570.65
7.00 (6.4–7.0) 6.8770.07 0.70 (0.2–1.2) 0.7470.11 14.2 (9.5–20.2) 14.4071.07 0.7 (0.2–1.2) 0.7270.11 145 (7.0–314) 144734.50 71.1(44.4–126.6) 7477.90 51.3 (7.2–83.9) 47.9077.87 509 (108–831) 509776.00 830 (179.3–2802) 9907406. 2.47 (0.62–3.66) 2.1870.36 540 (430–670) 540748 0.61 (0.24–0.95) 0.6070.09 1.71(0.34–4.66) 1.9270.62
Results are expressed as medians and range, means7SEM (standard error).
1000
480±30*
540±48
600±40*
SPO (U/100µL)
800 600 400 200 *p<0.05
convincing evidence that free radical mechanisms are involved in the pathogenesis of asthma and that antioxidants may be protective. Further studies involving larger groups comparing saliva, serum and BAL parameters should be conducted in order to fully understand the role of salivary antioxidants in children with asthma.
Asthma Attack Asthma Remission Healthy
0
Figure. 1 Comparison of SPO levels between healthy controls and asthmatic patients during attack and remission.
fluids such as induced sputum, BAL and blood. Furthermore, no specific inflammatory parameters were measured in addition to the clinical and spirometry evaluations. This pilot study shows that asthmatic patients have decreased levels of antioxidant enzyme activity in saliva as a result of the ongoing inflammatory and oxidative process. There is fairly
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