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Effects of storage time on stability of salivary immunoglobulin A and lysozyme
Clinica Chimica Acta 338 (2003) 131 – 134 www.elsevier.com/locate/clinchim
Effects of storage time on stability of salivary immunoglobulin A and lysozyme Vivian Ng, David Koh *, Qiuyun Fu, Sin-Eng Chia Department of Community, Occupational and Family Medicine, Faculty of Medicine (MD3), National University of Singapore, 16, Medical Drive, Singapore 117597, Singapore Received 10 July 2003; received in revised form 18 August 2003; accepted 18 August 2003
Abstract Background: In many research settings, storage of samples prior to analysis is unavoidable. This study investigates the effects of storage time on stability of salivary immunoglobulin A (IgA) and lysozyme. Methods: Saliva samples were obtained from 30 healthy adults. Each sample was divided into five aliquots and stored at 30 jC until analysis. The samples were measured for IgA and lysozyme concentrations after 1, 2, 3, 8 and 12 months of storage using enzyme-linked immunosorbent assay. Results: There was a decline in the concentrations of IgA and lysozyme with increasing storage time. Repeated measures analyses for both salivary IgA and lysozyme showed a significant difference after 8 months of storage as compared to the 1st month ( p < 0.05). IgA levels decreased significantly with % change in majority of the samples >10% after storage for 8 months or more. A similar pattern was observed for lysozyme levels with % change in majority of the samples >14% when the samples were assayed at 8th month and beyond (mean% change F S.D.>14%). Conclusion: Salivary IgA and lysozyme concentrations remain stable for up to 3 months when stored at 30 jC. These findings have important implications with regard to measurement validity of salivary biomarkers research. D 2003 Elsevier B.V. All rights reserved. Keywords: Salivary IgA; Salivary lysozyme; Stability; Salivary biomarkers; Storage duration
1. Introduction Saliva has increasingly been used as a diagnostic fluid in the last 10 years [1]. The use of saliva, rather than blood for diagnosis, has several advantages. A major advantage is that the collection of saliva is noninvasive, and generally much preferred by individuals. Thus, repeated sampling for serial measurement is * Corresponding author. Tel.: +65-6874-4988; fax: +65-67791489. E-mail address: [email protected] (D. Koh). 0009-8981/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.cccn.2003.08.012
possible. There is also a potential for cost saving as it does not require a trained phlebotomist to perform the collection of saliva. A wide range of salivary biomarkers can be measured at present. These include toxins (e.g., heavy metals such as Pb and Cd), hormones (e.g., cortisol and DHEA), various drugs and their metabolites (e.g., cotinine), and measures of mucosal function (e.g., immunoglobulins and enzymes) [2]. Salivary Immunoglobulin A (IgA) has an important role in mucosal immunity [3]. It is important in the protection against infection of upper respiratory infec-
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V. Ng et al. / Clinica Chimica Acta 338 (2003) 131–134
tion [4]. Habitual exercise, particularly intensive exercise, can cause a decrease of IgA in mucosal secretion which leads to the risk of respiratory illness [5]. However, regular and moderate exercise results in increased of IgA production, which may contribute to a decreased risk of infection [6]. Salivary IgA secretion rate has been shown to be sensitive to psychological variables [7,8]. Lysozyme is an enzyme which shows bacterial activity by splitting the bond between N-acetyl glucosamine and muramic acid in the mucopeptide components of the bacterial cell wall [9]. It is one of the major non-immunological antimicrobial proteins in saliva [10]. Salivary lysozyme has also been used as a stress biomarker [11,12]. Due to logistic, financial, practical and methodological reasons, it is not always possible to analyze samples immediately after collection. Thus, storage prior to samples analysis is necessary. This study investigates the stability of the salivary IgA and lysozyme when the samples were stored for 1, 2, 3, 8 and 12 months.
2. Materials and methods The study population consisted of 30 male workers employed in a factory manufacturing a polyvinyl chloride stabilizer. The National University Hospital Institutional Review Board, Singapore granted us approval to carry out this study and informed consent was obtained from the subjects.
and stored at 30 jC until analysis. All samples were stored without adding any preservatives. The samples were measured for IgA and lysozyme at after 1, 2, 3, 8 and 12 months of storage. Salivary IgA concentration (Ag/ml) was determined using the ELISA method [13,14]. The intraand inter-assay coefficient of variation (CV) were 5.1% and 9.8%, respectively, for a mean level of 170 Ag/ml. Salivary lysozyme concentration was measured using a competitive ELISA method that was developed in our laboratory, as described previously [12]. The intra- and inter-assay CV were 7.7% and 17.1%, respectively, mean 50.7 Ag/ml. All the samples were assayed in duplicate and the mean of the duplicate results was used as a final value. 2.2. Data analysis Data were analysed with SPSS software Base 11.0 (SPSS, Chicago USA). As the lysozyme data showed a positively skewed distribution, log transformation was performed to meet the assumption of normality and the transformed data were used for the subsequent statistical analyses. Within sample variation of different storage stages was evaluated using the Analysis for Variance for repeated measure design. A simple contrast was specified to compare the mean of each level with the mean of the 1st measurement. Alterations of sample concentrations exceeding F 2 intraassay CV of the assays were defined as denaturation [15]. These values were 10% for IgA and 14% for lysozyme.
2.1. Sample collection, preparation, storage and analysis Workers were advised not to smoke, eat or drink (except water) for 1 h prior to the saliva collection. After rinsing their mouths with water thoroughly, a single, unstimulated saliva sample, accumulated over 5 min was obtained. Immediately after collection, samples were kept in an ice box. Samples were transported to the laboratory within 2 h. All 30 saliva samples (with the volumes >2 ml) collected on the same day were used for this study. Once in the laboratory, samples were centrifuged for 15 min at 3500 rpm to remove any particles or sediments. Each sample was divided into five aliquots
Table 1 Mean and 95% confidence intervals for salivary IgA and lysozyme of 30 salivary samples at different storage times Storage time (month)
IgA concentration (Ag/ml)
Lysozyme concentration (Ag/ml)
Arithmetic mean
95% Confidence interval
Geometric mean
95% Confidence interval
1 2 3 8 12
218.9 218.2 216.4 203.4 193.0
195.9 – 241.8 193.9 – 242.6 193.0 – 239.8 174.3 – 232.4 163.6 – 222.5
249.8 245.1 243.7 210.2 196.6
182.8 – 341.3 179.5 – 334.7 178.6 – 332.8 151.9 – 290.9 140.8 – 283.5
V. Ng et al. / Clinica Chimica Acta 338 (2003) 131–134
3. Results 3.1. Salivary IgA After 1 month of storage, IgA concentration of the samples ranged from 124.3 to 333.5 Ag/ml. There was a decline in the concentrations of IgA with increasing storage time. The results of the subsequent analyses are shown in Table 1. Repeated measures analysis of variance with simple contrast showed a significant difference of salivary IgA levels at the 8th month of storage and beyond, as compared to the 1st month measurement ( p < 0.05). The variability of the samples also increased with increasing storage times. The
133
% change of majority of the samples for IgA concentrations at the 8th and 12th were >10% below the value at the 1st month (Fig. 1a). 3.2. Salivary lysozyme The concentration of lysozyme after 1 month of storage ranged from 59.7 to 1062.3 Ag/ml. Also, there was a decline in the concentrations of lysozyme with increasing storage time (Table 1). Within sample variation of repeated measures analyses also revealed that lysozyme concentrations decreased significantly after 8th and 12th months of storage as compared to 1st month measurement ( p < 0.05). The pattern of degradation for lysozyme levels was similar to IgA. The % change of majority of samples exceeded 14% when the samples were assayed at the 8th and 12th months (Fig. 1b).
4. Discussion As compared to plasma, saliva contains higher bacterial loads and higher levels of enzymes that may degrade the contents of proteins if it is not stored properly [2]. Salivary IgA and lysozyme are proteins that should be stored and handled carefully so as to avoid changes in the contents. Our findings indicate that salivary IgA and lysozyme concentrations are stable for up to 3 months when stored at 30 jC. Such findings have not been previously reported. Another study by Sitaramamma et al. [16] examined the effect of storage time on tear samples. They found that IgA and lysozyme in tear samples could be stored for 4 months at 70 jC without any significant reduction in the protein concentrations. This study indicated that salivary IgA and lysozyme concentrations remained stable for up to 3 months when stored at 30 jC. These findings have important implications with regards to measurement validity in the field of salivary biomarkers research. Fig. 1. Percent change of salivary IgA (a) and lysozyme (b) at different storage durations. (a) Salivary IgA—30 samples stored at 30 jC. The data points and error bars represent the mean F S.D.; the horizontal dotted lines indicate the acceptable range ( F 10%). (b) Salivary lysozyme—30 samples stored at 30 jC. The data points and error bars represent the mean F S.D.; the horizontal dotted lines indicate the acceptable range ( F 14%).
Acknowledgements The authors thank the management and the workers of Sun Ace Kakoh Pte. Ltd. for their participation
134
V. Ng et al. / Clinica Chimica Acta 338 (2003) 131–134
in this study. This study was supported by grant R186-000-056-305 from the Biomedical Research Council of Singapore.
References [1] Streckfus CF, Bigler LR. Saliva as a diagnostic fluid. Oral Dis 2002;8:69 – 76. [2] Malamud D, Tabak L. Saliva as a diagnostic fluid. Ann N Y Acad Sci 1993;694:1 – 348. [3] Mestecky J. Saliva as a manifestation of the common mucosal immune system. Ann N Y Acad Sci 1993;694:184 – 94. [4] Kugler J, Hess M, Haake D. Secretion of salivary immunoglobulin A in relation to age, saliva flow, mood states, secretion of albumin, cortisol, and catecholamines in saliva. J Clin Immunol 1992;12:45 – 9. [5] Gleeson M, Pyne DB. Special feature for the Olympics: effects of exercise on the immune system: exercise effects on mucosal immunity. Immunol Cell Biol 2000;78:536 – 44. [6] Klentrou P, Cieslak T, MacNeil M, Vintinner A, Plyley M. Effect of moderate exercise on salivary immunoglobulin A and infection risk in humans. Eur J Appl Physiol 2002;87:153 – 8. [7] Jemmott III JB, Borysenko JZ, Borysenko M, McClelland DC, Chapman R, Meyer D, et al. Academic stress, power motivation, and decrease in secretion rate of salivary secretory immunoglobulin A. Lancet 1983;1:1400 – 2.
[8] Miletic ID, Schiffman SS, Miletic VD, Sattely-Miller EA. Salivary IgA secretion rate in young and elderly persons. Physiol Behav 1996;60:243 – 8. [9] Schenkels LC, Veerman EC, Nieuw Amerongen AV. Biochemical composition of human saliva in relation to other mucosal fluids. Crit Rev Oral Biol Med 1995;6:161 – 75. [10] Yeh CK, Dodds MW, Zuo P, Johnson DA. A population-based study of salivary lysozyme concentrations and candidal counts. Arch Oral Biol 1997;42:25 – 31. [11] Perera S, Uddin M, Hayes JA. Salivary lysozyme: a noninvasive marker for the study of the effects of stress on natural immunity. Int J Behav Med 1997;4:170 – 8. [12] Yang Y, Koh D, Ng V, Lee CY, Goh SH, Anantharaman V, et al. Self-perceived work-related stress and the relation with salivary IgA and lysozyme among emergency department nurses. Occup Environ Med 2002;59:836 – 41. [13] Henningsen GM, Hurrell Jr JJ, Baker F, Douglas C, MacKenzie BA, Robertson SK, et al. Measurement of salivary immunoglobulin A as an immunologic biomarker of job stress. Scand J Work Environ Health 1992;18:133 – 6. [14] Miletic ID, Schiffman SS, Miletic VD, Sattely-Miller EA. Salivary IgA secretion rate in young and elderly persons. Physiol Behav 1996 (Jul);60(1):243 – 8. [15] Groschl M, Wagner R, Rauh M, Dorr HG. Stability of salivary steroids: the influences of storage, food and dental care. Steroids 2001;66(10):737 – 41. [16] Sitaramamma T, Shivaji S, Rao GN. Effect of storage on protein concentration of tear samples. Curr Eye Res 1998; 17(10):1027 – 35.
Effects of storage time on stability of salivary immunoglobulin A and lysozyme Vivian Ng, David Koh *, Qiuyun Fu, Sin-Eng Chia Department of Community, Occupational and Family Medicine, Faculty of Medicine (MD3), National University of Singapore, 16, Medical Drive, Singapore 117597, Singapore Received 10 July 2003; received in revised form 18 August 2003; accepted 18 August 2003
Abstract Background: In many research settings, storage of samples prior to analysis is unavoidable. This study investigates the effects of storage time on stability of salivary immunoglobulin A (IgA) and lysozyme. Methods: Saliva samples were obtained from 30 healthy adults. Each sample was divided into five aliquots and stored at 30 jC until analysis. The samples were measured for IgA and lysozyme concentrations after 1, 2, 3, 8 and 12 months of storage using enzyme-linked immunosorbent assay. Results: There was a decline in the concentrations of IgA and lysozyme with increasing storage time. Repeated measures analyses for both salivary IgA and lysozyme showed a significant difference after 8 months of storage as compared to the 1st month ( p < 0.05). IgA levels decreased significantly with % change in majority of the samples >10% after storage for 8 months or more. A similar pattern was observed for lysozyme levels with % change in majority of the samples >14% when the samples were assayed at 8th month and beyond (mean% change F S.D.>14%). Conclusion: Salivary IgA and lysozyme concentrations remain stable for up to 3 months when stored at 30 jC. These findings have important implications with regard to measurement validity of salivary biomarkers research. D 2003 Elsevier B.V. All rights reserved. Keywords: Salivary IgA; Salivary lysozyme; Stability; Salivary biomarkers; Storage duration
1. Introduction Saliva has increasingly been used as a diagnostic fluid in the last 10 years [1]. The use of saliva, rather than blood for diagnosis, has several advantages. A major advantage is that the collection of saliva is noninvasive, and generally much preferred by individuals. Thus, repeated sampling for serial measurement is * Corresponding author. Tel.: +65-6874-4988; fax: +65-67791489. E-mail address: [email protected] (D. Koh). 0009-8981/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.cccn.2003.08.012
possible. There is also a potential for cost saving as it does not require a trained phlebotomist to perform the collection of saliva. A wide range of salivary biomarkers can be measured at present. These include toxins (e.g., heavy metals such as Pb and Cd), hormones (e.g., cortisol and DHEA), various drugs and their metabolites (e.g., cotinine), and measures of mucosal function (e.g., immunoglobulins and enzymes) [2]. Salivary Immunoglobulin A (IgA) has an important role in mucosal immunity [3]. It is important in the protection against infection of upper respiratory infec-
132
V. Ng et al. / Clinica Chimica Acta 338 (2003) 131–134
tion [4]. Habitual exercise, particularly intensive exercise, can cause a decrease of IgA in mucosal secretion which leads to the risk of respiratory illness [5]. However, regular and moderate exercise results in increased of IgA production, which may contribute to a decreased risk of infection [6]. Salivary IgA secretion rate has been shown to be sensitive to psychological variables [7,8]. Lysozyme is an enzyme which shows bacterial activity by splitting the bond between N-acetyl glucosamine and muramic acid in the mucopeptide components of the bacterial cell wall [9]. It is one of the major non-immunological antimicrobial proteins in saliva [10]. Salivary lysozyme has also been used as a stress biomarker [11,12]. Due to logistic, financial, practical and methodological reasons, it is not always possible to analyze samples immediately after collection. Thus, storage prior to samples analysis is necessary. This study investigates the stability of the salivary IgA and lysozyme when the samples were stored for 1, 2, 3, 8 and 12 months.
2. Materials and methods The study population consisted of 30 male workers employed in a factory manufacturing a polyvinyl chloride stabilizer. The National University Hospital Institutional Review Board, Singapore granted us approval to carry out this study and informed consent was obtained from the subjects.
and stored at 30 jC until analysis. All samples were stored without adding any preservatives. The samples were measured for IgA and lysozyme at after 1, 2, 3, 8 and 12 months of storage. Salivary IgA concentration (Ag/ml) was determined using the ELISA method [13,14]. The intraand inter-assay coefficient of variation (CV) were 5.1% and 9.8%, respectively, for a mean level of 170 Ag/ml. Salivary lysozyme concentration was measured using a competitive ELISA method that was developed in our laboratory, as described previously [12]. The intra- and inter-assay CV were 7.7% and 17.1%, respectively, mean 50.7 Ag/ml. All the samples were assayed in duplicate and the mean of the duplicate results was used as a final value. 2.2. Data analysis Data were analysed with SPSS software Base 11.0 (SPSS, Chicago USA). As the lysozyme data showed a positively skewed distribution, log transformation was performed to meet the assumption of normality and the transformed data were used for the subsequent statistical analyses. Within sample variation of different storage stages was evaluated using the Analysis for Variance for repeated measure design. A simple contrast was specified to compare the mean of each level with the mean of the 1st measurement. Alterations of sample concentrations exceeding F 2 intraassay CV of the assays were defined as denaturation [15]. These values were 10% for IgA and 14% for lysozyme.
2.1. Sample collection, preparation, storage and analysis Workers were advised not to smoke, eat or drink (except water) for 1 h prior to the saliva collection. After rinsing their mouths with water thoroughly, a single, unstimulated saliva sample, accumulated over 5 min was obtained. Immediately after collection, samples were kept in an ice box. Samples were transported to the laboratory within 2 h. All 30 saliva samples (with the volumes >2 ml) collected on the same day were used for this study. Once in the laboratory, samples were centrifuged for 15 min at 3500 rpm to remove any particles or sediments. Each sample was divided into five aliquots
Table 1 Mean and 95% confidence intervals for salivary IgA and lysozyme of 30 salivary samples at different storage times Storage time (month)
IgA concentration (Ag/ml)
Lysozyme concentration (Ag/ml)
Arithmetic mean
95% Confidence interval
Geometric mean
95% Confidence interval
1 2 3 8 12
218.9 218.2 216.4 203.4 193.0
195.9 – 241.8 193.9 – 242.6 193.0 – 239.8 174.3 – 232.4 163.6 – 222.5
249.8 245.1 243.7 210.2 196.6
182.8 – 341.3 179.5 – 334.7 178.6 – 332.8 151.9 – 290.9 140.8 – 283.5
V. Ng et al. / Clinica Chimica Acta 338 (2003) 131–134
3. Results 3.1. Salivary IgA After 1 month of storage, IgA concentration of the samples ranged from 124.3 to 333.5 Ag/ml. There was a decline in the concentrations of IgA with increasing storage time. The results of the subsequent analyses are shown in Table 1. Repeated measures analysis of variance with simple contrast showed a significant difference of salivary IgA levels at the 8th month of storage and beyond, as compared to the 1st month measurement ( p < 0.05). The variability of the samples also increased with increasing storage times. The
133
% change of majority of the samples for IgA concentrations at the 8th and 12th were >10% below the value at the 1st month (Fig. 1a). 3.2. Salivary lysozyme The concentration of lysozyme after 1 month of storage ranged from 59.7 to 1062.3 Ag/ml. Also, there was a decline in the concentrations of lysozyme with increasing storage time (Table 1). Within sample variation of repeated measures analyses also revealed that lysozyme concentrations decreased significantly after 8th and 12th months of storage as compared to 1st month measurement ( p < 0.05). The pattern of degradation for lysozyme levels was similar to IgA. The % change of majority of samples exceeded 14% when the samples were assayed at the 8th and 12th months (Fig. 1b).
4. Discussion As compared to plasma, saliva contains higher bacterial loads and higher levels of enzymes that may degrade the contents of proteins if it is not stored properly [2]. Salivary IgA and lysozyme are proteins that should be stored and handled carefully so as to avoid changes in the contents. Our findings indicate that salivary IgA and lysozyme concentrations are stable for up to 3 months when stored at 30 jC. Such findings have not been previously reported. Another study by Sitaramamma et al. [16] examined the effect of storage time on tear samples. They found that IgA and lysozyme in tear samples could be stored for 4 months at 70 jC without any significant reduction in the protein concentrations. This study indicated that salivary IgA and lysozyme concentrations remained stable for up to 3 months when stored at 30 jC. These findings have important implications with regards to measurement validity in the field of salivary biomarkers research. Fig. 1. Percent change of salivary IgA (a) and lysozyme (b) at different storage durations. (a) Salivary IgA—30 samples stored at 30 jC. The data points and error bars represent the mean F S.D.; the horizontal dotted lines indicate the acceptable range ( F 10%). (b) Salivary lysozyme—30 samples stored at 30 jC. The data points and error bars represent the mean F S.D.; the horizontal dotted lines indicate the acceptable range ( F 14%).
Acknowledgements The authors thank the management and the workers of Sun Ace Kakoh Pte. Ltd. for their participation
134
V. Ng et al. / Clinica Chimica Acta 338 (2003) 131–134
in this study. This study was supported by grant R186-000-056-305 from the Biomedical Research Council of Singapore.
References [1] Streckfus CF, Bigler LR. Saliva as a diagnostic fluid. Oral Dis 2002;8:69 – 76. [2] Malamud D, Tabak L. Saliva as a diagnostic fluid. Ann N Y Acad Sci 1993;694:1 – 348. [3] Mestecky J. Saliva as a manifestation of the common mucosal immune system. Ann N Y Acad Sci 1993;694:184 – 94. [4] Kugler J, Hess M, Haake D. Secretion of salivary immunoglobulin A in relation to age, saliva flow, mood states, secretion of albumin, cortisol, and catecholamines in saliva. J Clin Immunol 1992;12:45 – 9. [5] Gleeson M, Pyne DB. Special feature for the Olympics: effects of exercise on the immune system: exercise effects on mucosal immunity. Immunol Cell Biol 2000;78:536 – 44. [6] Klentrou P, Cieslak T, MacNeil M, Vintinner A, Plyley M. Effect of moderate exercise on salivary immunoglobulin A and infection risk in humans. Eur J Appl Physiol 2002;87:153 – 8. [7] Jemmott III JB, Borysenko JZ, Borysenko M, McClelland DC, Chapman R, Meyer D, et al. Academic stress, power motivation, and decrease in secretion rate of salivary secretory immunoglobulin A. Lancet 1983;1:1400 – 2.
[8] Miletic ID, Schiffman SS, Miletic VD, Sattely-Miller EA. Salivary IgA secretion rate in young and elderly persons. Physiol Behav 1996;60:243 – 8. [9] Schenkels LC, Veerman EC, Nieuw Amerongen AV. Biochemical composition of human saliva in relation to other mucosal fluids. Crit Rev Oral Biol Med 1995;6:161 – 75. [10] Yeh CK, Dodds MW, Zuo P, Johnson DA. A population-based study of salivary lysozyme concentrations and candidal counts. Arch Oral Biol 1997;42:25 – 31. [11] Perera S, Uddin M, Hayes JA. Salivary lysozyme: a noninvasive marker for the study of the effects of stress on natural immunity. Int J Behav Med 1997;4:170 – 8. [12] Yang Y, Koh D, Ng V, Lee CY, Goh SH, Anantharaman V, et al. Self-perceived work-related stress and the relation with salivary IgA and lysozyme among emergency department nurses. Occup Environ Med 2002;59:836 – 41. [13] Henningsen GM, Hurrell Jr JJ, Baker F, Douglas C, MacKenzie BA, Robertson SK, et al. Measurement of salivary immunoglobulin A as an immunologic biomarker of job stress. Scand J Work Environ Health 1992;18:133 – 6. [14] Miletic ID, Schiffman SS, Miletic VD, Sattely-Miller EA. Salivary IgA secretion rate in young and elderly persons. Physiol Behav 1996 (Jul);60(1):243 – 8. [15] Groschl M, Wagner R, Rauh M, Dorr HG. Stability of salivary steroids: the influences of storage, food and dental care. Steroids 2001;66(10):737 – 41. [16] Sitaramamma T, Shivaji S, Rao GN. Effect of storage on protein concentration of tear samples. Curr Eye Res 1998; 17(10):1027 – 35.