- Email: [email protected]
Salivary osmolality in individuals with cerebral palsy
archives of oral biology 55 (2010) 855–860
available at www.sciencedirect.com
journal homepage: http://www.elsevier.com/locate/aob
Salivary osmolality in individuals with cerebral palsy Maria Teresa B. Santos a, Renata O. Guare´ b, Mariana F. Leite b,*, Maria Cristina D. Ferreira b, Marcelino S. Dura˜o c, Jose R. Jardim d a
Individuals with Special Needs, Universidade Cruzeiro do Sul, Director Dentistry Division Lar Escola Sa˜o Francisco, Sa˜o Paulo, Brazil Centro de Cieˆncias Biolo´gicas e da Sau´de, Universidade Cruzeiro do Sul, Sa˜o Paulo, Brazil c Nephrology Division, Universidade Federal de Sa˜o Paulo, Brazil d Respiratory Diseases, Universidade Federal de Sa˜o, Brazil b
article info
abstract
Article history:
Objective: To measure the salivary flow rate, osmolality, electrolyte and total protein con-
Accepted 28 July 2010
centrations in individuals with cerebral palsy (CP). Design: Thirty-eight individuals with CP were divided according to the neuromotor abnor-
Keywords:
mality type (total, spastic and dyskinectic) and compared to 22 nondisabled children (control
Salivary flow rate
group). Whole saliva was collected under slight suction. The salivary parameters studied
Salivary osmolality
were salivary flow rate, osmolality, sodium, potassium, chloride and total protein concen-
Total protein concentration
trations.
Salivary electrolytes
Results: CP individuals, with both neuromotor abnormality types (spastic and dyskinectic),
Cerebral palsy
presented an increase in salivary osmolality, total protein, potassium and chloride concentrations compared to the control group ( p < 0.05). Moreover, a reduction in salivary flow rate was verified in spastic individuals ( p < 0.05). Conclusion: The reduction in salivary flow rate and increase in osmolality, total protein and electrolyte concentrations of saliva from cerebral palsy individuals could be caused by hypohydration status. # 2010 Elsevier Ltd. All rights reserved.
1.
Introduction
Cerebral palsy (CP) describes a group of permanent disorders that involves movement and posture development causing activity limitation, which are attributed to nonprogressive disturbances occurring in the developing fetal or infant brain. CP motor disorders are often accompanied by epilepsy, secondary musculoskeletal problems and disturbances of sensation, perception, cognition, communication and behavior.1 This condition is the most common cause of severe physical disability in childhood,2 with an estimated prevalence of 2.4 per 1000 children.3 Previous studies reported alterations in salivary flow rate and biochemical parameters of whole saliva in CP indivi-
duals.4–7 The variation in sodium and potassium concentrations suggests some injury in the energy-dependent reabsorption of electrolytes in the duct system of salivary gland is associated with CP.4 A reduction in digestive and antimicrobial enzymes activities, amylase and peroxidase, respectively, have also been described in CP individuals.6 Increased sialic acid concentrations could be related to increased saliva viscosity in these individuals.6 Delayed recovery of salivary pH, involving compromised ability to buffer exogenous acid after using certain medications was also observed in CP individuals.5 Xerostomia has been described in cerebral palsy individuals submitted to drug therapy, surgery and botulinum toxin injections, as a form of drooling management.8
* Corresponding author at: Cieˆncias Biolo´gicas e da Sau´de – CBS, Universidade Cruzeiro do Sul, Rua Aure´lia, 1821, apto121 – Vila Romana, Sa˜o Paulo, SP, CEP 05046-001, Brazil. Tel.: +55 11 78727095. E-mail address: [email protected] (M.F. Leite). 0003–9969/$ – see front matter # 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.archoralbio.2010.07.016
856
archives of oral biology 55 (2010) 855–860
The organic and inorganic composition of saliva has several important biological functions. The use of saliva, crevicular fluid and mucosal transudates for drug monitoring and to detect several oral and systemic diseases has been described, particularly in autoimmune disorders, cardiovascular diseases, endocrinology, infectious disease, nephrology, oncology and psychiatry.9,10 The secretion of saliva and electrolytes is stimulated by the action of neurotransmitters of the autonomic nervous system. The concentration of salivary electrolytes depends on the action of co-transporters and membrane regulatory proteins, such as aquaporins,11,12 and on the action of the salivary duct system. The increase in intracellular concentration of ions Cl , due to the action of Na+–K+–2Cl and Cl – HCO3 channels, is responsible for an osmotic gradient that facilitates the secretion of potassium, sodium, bicarbonate and others salivary electrolytes.13 Saliva composition can vary according to salivary flow rate, the type of gland and hydration status.14 Water is the medium of circulatory function, biochemical reactions, metabolism, and substrate transport across cellular membranes, temperature regulation and numerous other physiological processes.15 Fluid-electrolyte turnover and whole-body water balance change constantly. Several methods have been used to measure the state of hydration in the body, including plasma osmolality, bioelectrical impedance spectroscopy, body mass change, urine tests and others.15 Osmolality is the concentration of a solution expressed in milliosmoles of solute particles per kilogram of water. It has been suggested that decreased levels of hydration (dehydration) may cause diminished salivary output.16 Some studies have shown that the salivary flow rate, total protein concentration and osmolality of saliva may be potential noninvasive markers of whole-body hydration status during intentionally progressive acute dehydration in healthy subjects.17 Moreover, theses parameters are strongly correlated with plasma osmolality (a widely accepted hydratation index), demonstrating that changes in saliva components reflect changes in hydratation status during hypertonichypovolemia.17 It has been clearly shown that a reduced salivary flow rate occurs in CP individuals.4–7 Reduced salivary flow rate could be either hyperosmolar or isosmolar. The measurement of osmolality, protein concentration and electrolytes could provide information regarding the real condition of salivary osmolality. To the best of our knowledge, no report regarding salivary osmolality status in individuals with CP has been published. The aim of this study was to measure the salivary flow rate, osmolality, electrolytes and total protein concentration in cerebral palsy individuals.
2.
Material and methods
2.1.
Subjects
This project was reviewed by the Human Research Ethics Committee and approved by the Federal University of Sao Paulo Institutional Review Board (IRB) and granted under protocol approval 1034/08. After being informed of the aim
of the investigation, written informed consent for participation and publication was obtained from the adult responsible for each child/individual who agreed to participate in the study. A group of 38 noninstitutionalized male and female children (aged 7–14 years-old) with CP, attending the Special Children’s Elementary School of Lar Escola Sao Francisco, Rehabilitation Center/Unifesp, were enrolled in this study. The inclusion criterion was a medical diagnosis of CP and the exclusion criteria were the use of any drugs that interfere with saliva secretion (anticholinergic and neuroleptic drugs, benzodiazepines) for at least 72 h prior to examination, a history of head and neck radiation and surgical procedures to reduce drooling. Patient medical records were reviewed for clinical data concerning the type of movement disorder (spasticity and dyskinesia). The control group consisted of 22 nondisabled children (aged 6–14 years-old) and the primary inclusion criterion was absence of neurological damage. None of the participants reported any complaints suggestive of salivary-gland dysfunction or were using any medication that could affect salivary secretion.
2.2.
Saliva collection
At least 2 h after the previous meal, unstimulated whole saliva was collected between 8 and 10 a.m., to minimise the circadian rhythm effects, using slight suction through a soft plastic catheter. Saliva produced in the first 10 s was discarded and the subsequent saliva was collected for exactly 5 min in a graduated cylinder to calculate the initial flow rate (mL/min). During the collection period, all individuals remained comfortably seated in a ventilated and illuminated schoolroom. If a child did not permit saliva collection, he or she was excluded. Soon after collection, saliva was frozen in dry ice, transported to the laboratory and stored at 80 8C until analysis.
2.3.
Measurement of salivary parameters
Salivary osmolality was measured using a freezing point depression Osmometer (Model Wide-Range Osmometer 3W2, Advanced Instruments, Massachusetts, USA). The sodium, potassium and chloride concentrations were measured using the respective Ion Selective Electrodes (AU400 Chemistry Analyzer, Olympus, Shizuoka-zen, Japan). Salivary total protein concentration was estimated by the Bradford method18 using bovine serum albumin as a standard.
2.4.
Statistical analysis
The data are presented as mean standard deviation (SD). Biochemical parameters of the groups studied were compared by Analysis of Variance and the Tukey test. The level of significance adopted was 5% ( p < 0.05).
3.
Results
Figs. 1–6 show the salivary parameters of healthy and CP individuals according to the neuromotor abnormality type
archives of oral biology 55 (2010) 855–860
[(Fig._1)TD$IG]
857
Fig. 1 – Salivary flow rate (mL/min) of healthy (control) (n = 22), total cerebral palsy (total PC) (n = 38), spastic (n = 29), and dyskinectic (n = 9) individuals. The data are expressed as mean W SD (columns and lines, respectively). Statistical differences compared to the control group are represented by asterisks (*) ( p < 0.05).
[(Fig._2)TD$IG]
Fig. 2 – Total protein concentration (mL/min) of healthy (control) (n = 22), total cerebral palsy (total PC) (n = 38), spastic (n = 29), and dyskinectic (n = 9) individuals. The data are expressed as mean W SD (columns and lines, respectively). Statistical differences compared to the control group are represented by asterisks (*) ( p < 0.05).
(total CP, spastic, and dyskinectic). The salivary parameters studied were osmolality, sodium, potassium, chloride and total protein concentrations and salivary flow rate. The results were expressed as mean SD. CP individuals with neuromotor abnormality type (total, spastic and dyskinectic) presented an increase in salivary osmolality (25%, 25% and 28%, respectively) (Fig. 3), total protein concentrations (14%, 14% and 19%, respectively) (Fig. 2), potassium (25%, 26% and 28%, respectively) (Fig. 5) and chloride (17% and 18% in the total CP and spastic, respectively) (Fig. 6) concentrations compared to the control group ( p < 0.05). Moreover, a reduction in salivary flow rate of total CP (29%) and spastic individuals (32%) was verified compared to the control group ( p < 0.05) (Fig. 1). No changes were observed in the sodium concentrations of the groups studied (Fig. 4).
4.
Discussion
Cerebral palsy is a disease which affects the motor functions of the individual and presents a series of oral manifestations, both clinical and biochemical. This study shows for the first time that individuals with CP present salivary changes characterised by an increase in osmolality, total protein, potassium and chloride concentrations. Moreover, the reduction in salivary flow rate in CP individuals observed in this study is in agreement with previous reports4–7 and supports the idea of hyperconcentrated saliva, which is more common in dehydrated status. Widely accepted and frequently used markers of hydration status include hematological and urinary parameters; however, the use of salivary parameters as indication of water loss have also been proposed.17,19 These studies showed that the
858
archives of oral biology 55 (2010) 855–860
[(Fig._3)TD$IG]
Fig. 3 – Salivary osmolality (mOsm/Kg H2O) of healthy (control) (n = 22), total cerebral palsy (total PC) (n = 38), spastic (n = 29), and dyskinectic (n = 9) individuals. The data are expressed as mean W SD (columns and lines, respectively). Statistical differences compared to the control group are represented by asterisks (*) ( p < 0.05).
[(Fig._4)TD$IG]
Fig. 4 – Sodium concentration (mEq/L) of healthy (control) (n = 22), total cerebral palsy (total PC) (n = 38), spastic (n = 29), and dyskinectic (n = 9) individuals. The data are expressed as mean W SD (columns and lines, respectively).
[(Fig._5)TD$IG]
Fig. 5 – Potassium concentration (mEq/L) of healthy (control) (n = 22), total cerebral palsy (total PC) (n = 38), spastic (n = 29), and dyskinectic (n = 9) individuals. The data are expressed as mean W SD (columns and lines, respectively). Statistical differences compared to the control group are represented by asterisks (*) ( p < 0.05).
archives of oral biology 55 (2010) 855–860
[(Fig._6)TD$IG]
859
Fig. 6 – Chloride concentration (mEq/L) of healthy (control) (n = 22), total cerebral palsy (total PC) (n = 38), spastic (n = 29), and dyskinectic (n = 9) individuals. The data are expressed as mean W SD (columns and lines, respectively). Statistical differences compared to the control group are represented by asterisks (*) ( p < 0.05).
reduction in salivary flow rate and increase in osmolality and total protein concentration in the saliva of individuals subjected to procedures that lead to loss of water are correlated with other parameters of body dehydration. The individuals with CP who participated in this study presented similar results to individuals subjected to conditions of dehydration. The individuals of this study presented an increase in the concentration of electrolytes, i.e. potassium and chloride. These changes corroborate a previous study4 that suggested compromise of the duct system of salivary glands in individuals with CP. While passing through the duct system, the primary saliva secreted in the acini of the salivary glands undergoes reabsorption of sodium and chloride and excretion of potassium and bicarbonate.13 The submandibular gland undergoes changes in its secretory capacity, largely paracellular, according to the degree of osmolality of the medium.11 These changes are mediated by an osmosensor system in the basal membrane of the salivary gland.11 The hypohydration status probably causes an osmotic imbalance that alters the functions performed by the ducts of the salivary glands. Analysis of the results of this study suggests that individuals with CP present reduced salivary flow and changes in saliva composition due to lower water intake. The development of hypohydration in these individuals is due to the fact that they are mainly dependent on the initiative of being offering liquids by their caregivers. In addition, they do not complain of thirst and can hardly drink water by themselves. Moreover, CP compromises the ability of feeding skills,20 making it difficult to swallow all the water that is offered. Thus, those involved in the health care of individuals with CP should consider alternatives that would improve the hydration status of those under their care. The submandibular glands are the major contributors to resting mucous and serous saliva (unstimulated), while the parotid glands contribute to stimulated serous saliva.21 While the submandibular has a low oxygen demand, the parotid glands present high aerobic metabolism22 and therefore, are more prone to oxidative damage caused by reactive oxygen species (ROS). Water stress increases the formation of ROS resulting in lipid peroxidation, uncontrolled oxidation of thiol groups, denaturation of proteins and nucleic acid damage with severe consequences for overall metabolism. Other injuries
caused by hypohydration include reductions in cytoplasmatic and intracellular transport, alterations in cytoplasmic pH and cell membranes become more susceptible to attack by ROS.23 Unstimulated whole saliva was collected by slight suction without any difficulty.24,25 Previous studies reported alterations in salivary flow rate and organic and inorganic parameters of whole saliva in individuals with CP.4–6 The alterations observed in salivary total protein and electrolyte concentrations of individuals with CP could be caused by impaired functioning of the salivary glands, especially the submandibular glands. Thus, the results of this study lead to the conclusion that the diminished salivary flow rate and increased osmolality, total protein and electrolyte concentrations of saliva from individuals with cerebral palsy could be associated with hypohydration status. Moreover, hypohydration could be involved in possible oxidative damage in the salivary glands of individuals with cerebral palsy, further affecting the secretion of saliva.
Acknowledgment This study was supported by the Fundac¸a˜o de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) under protocol number 08/00960-6. Funding: This study was supported by the Fundac¸a˜o de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) under protocol number 08/00960-6, without any influence. Competing interests: There is nothing to declare. Ethical approval: This project was reviewed by the Human Research Ethics Committee and approved by the Federal University of Sao Paulo Institutional Review Board (IRB) and granted under protocol approval 1034/08.
references
1. Bax M, Goldstein M, Rosenbaum P, Leviton A, Paneth N, Dan B, et al. Executive committee for the definition of cerebral palsy. Proposed definition and classification of cerebral palsy. Dev Med Child Neurol 2005;47(8):571–6.
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archives of oral biology 55 (2010) 855–860
2. Kuban KC, Leviton A. Medical progress: cerebral palsy. N Engl Med 1994;330:188–95. 3. Hirtz D, Thurman DJ, Gwinn-Hardy K, Mohamed M, Chaudhuri AR, Zalutsky R. How common are the ‘‘common’’ neurologic disorders? Neurology 2007;68:326–37. 4. Siqueira WL, Rodrigues Santos MT, De Oliveira E, Nicolau J. Comparison of electrolyte concentrations in whole saliva of individuals with and without cerebral palsy. Quintessence Int 2007;38(4):301–6. 5. Siqueira WL, Santos MT, Elangovan S, Simoes A, Nicolau J. The influence of valproic acid on salivary pH in children with cerebral palsy. Spec Care Dentist 2007;27(2):64–6. 6. Rodrigues Santos MT, Siqueira WL, Nicolau J. Amylase and peroxidase activities and sialic acid concentration in saliva of adolescents with cerebral palsy. Quintessence Int 2007;38(6):467–72. 7. Santos MT, Masiero D, Simionato MR. Risk factors for dental caries in children with cerebral palsy. Spec Care Dentist 2002;22(3):103–7. 8. Scully C, Limeres J, Gleeson M, Toma´s I, Diz Drooling P. J Oral Pathol Med 2009;38(4):321–7. 9. Streckfus CF, Bigler LR. Saliva as a diagnose fluid. Oral Dis 2002;8:69–76. 10. Schipper RG, Silletti E, Vingerhoeds MH. Saliva as research material: biochemical, physicochemical and practical aspects. Arch Oral Biol 2007;52(12):1114–35. 11. Murakami M, Murdiastuti K, Hosoi K, Hill AE. AQP and the control of fluid transport in a salivary gland. J Membr Biol 2006;210(2):91–103. 12. Proctor GB, Carpenter GH. Regulation of salivary gland function by autonomic nerves. Auton Neurosci 2007;133 (1):3–18. 13. Nauntofte B. Regulation of electrolyte and fluid secretion in salivary acinar cells. Am J Physiol 1992;263(6 Pt 1):823–37. 14. Almeida PV, Gre´gio AM, Machado MA, de Lima AA, Azevedo LR. Saliva composition and functions: a comprehensive review. J Contemp Dent Pract 2008;9(3):72–80.
15. Armstrong LE. Assessing hydration status: the elusive gold standard. J Am Coll Nutr 2007;26(5):575S–84S. 16. Ship JA, Fischer DJ. The relationship between dehydration and parotid salivary gland function in young and older healthy adults. J Gerontol A Biol Sci Med Sci 1997;52(2): M310–9. 17. Walsh NP, Laing SJ, Oliver SJ, Montague JC, Walters R, Bilzon JL. Saliva parameters as potential indices of hydration status during acute dehydration. Med Sci Sports Exerc 2004;36(9):1535–42. 18. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 1976;72: 248–54. 19. Walsh NP, Montague JC, Callow N, Rowlands AV. Saliva flow rate, total protein concentration and osmolality as potential markers of whole body hydration status during progressive acute dehydration in humans. Arch Oral Biol 2004;49(2): 149–54. 20. dos Santos MT, Nogueira ML. Infantile reflexes and their effects on dental caries and oral hygiene in cerebral palsy individuals. J Oral Rehabil 2005;32(12):880–5. 21. Navazesh M, Kumar SK. Measuring salivary flow: challenges and opportunities. J Am Dent Assoc 2008;139:35S–40S. 22. Nicolau J, Sassaki KT. Metabolism of carbohydrate in the major salivary glands of the rats. Arch Oral Biol 1976;21: 659–61. 23. Franc¸a MB, Panek AD, Eleutherio EC. Oxidative stress and its effects during dehydration. Comp Biochem Physiol A Mol Integr Physiol 2007;146(4):621–31. 24. Siqueira WL, Siqueira MF, Mustacchi Z, de Oliveira E, Nicolau J. Salivary parameters in infants aged 12 to 60 months with Down syndrome. Spec Care Dentist 2007;27(5):202–5. 25. Bassoukou IH, Nicolau J, dos Santos MT. Saliva flow rate, buffer capacity, and pH of autistic individuals. Clin Oral Invest 2009;13(1):23–7.
available at www.sciencedirect.com
journal homepage: http://www.elsevier.com/locate/aob
Salivary osmolality in individuals with cerebral palsy Maria Teresa B. Santos a, Renata O. Guare´ b, Mariana F. Leite b,*, Maria Cristina D. Ferreira b, Marcelino S. Dura˜o c, Jose R. Jardim d a
Individuals with Special Needs, Universidade Cruzeiro do Sul, Director Dentistry Division Lar Escola Sa˜o Francisco, Sa˜o Paulo, Brazil Centro de Cieˆncias Biolo´gicas e da Sau´de, Universidade Cruzeiro do Sul, Sa˜o Paulo, Brazil c Nephrology Division, Universidade Federal de Sa˜o Paulo, Brazil d Respiratory Diseases, Universidade Federal de Sa˜o, Brazil b
article info
abstract
Article history:
Objective: To measure the salivary flow rate, osmolality, electrolyte and total protein con-
Accepted 28 July 2010
centrations in individuals with cerebral palsy (CP). Design: Thirty-eight individuals with CP were divided according to the neuromotor abnor-
Keywords:
mality type (total, spastic and dyskinectic) and compared to 22 nondisabled children (control
Salivary flow rate
group). Whole saliva was collected under slight suction. The salivary parameters studied
Salivary osmolality
were salivary flow rate, osmolality, sodium, potassium, chloride and total protein concen-
Total protein concentration
trations.
Salivary electrolytes
Results: CP individuals, with both neuromotor abnormality types (spastic and dyskinectic),
Cerebral palsy
presented an increase in salivary osmolality, total protein, potassium and chloride concentrations compared to the control group ( p < 0.05). Moreover, a reduction in salivary flow rate was verified in spastic individuals ( p < 0.05). Conclusion: The reduction in salivary flow rate and increase in osmolality, total protein and electrolyte concentrations of saliva from cerebral palsy individuals could be caused by hypohydration status. # 2010 Elsevier Ltd. All rights reserved.
1.
Introduction
Cerebral palsy (CP) describes a group of permanent disorders that involves movement and posture development causing activity limitation, which are attributed to nonprogressive disturbances occurring in the developing fetal or infant brain. CP motor disorders are often accompanied by epilepsy, secondary musculoskeletal problems and disturbances of sensation, perception, cognition, communication and behavior.1 This condition is the most common cause of severe physical disability in childhood,2 with an estimated prevalence of 2.4 per 1000 children.3 Previous studies reported alterations in salivary flow rate and biochemical parameters of whole saliva in CP indivi-
duals.4–7 The variation in sodium and potassium concentrations suggests some injury in the energy-dependent reabsorption of electrolytes in the duct system of salivary gland is associated with CP.4 A reduction in digestive and antimicrobial enzymes activities, amylase and peroxidase, respectively, have also been described in CP individuals.6 Increased sialic acid concentrations could be related to increased saliva viscosity in these individuals.6 Delayed recovery of salivary pH, involving compromised ability to buffer exogenous acid after using certain medications was also observed in CP individuals.5 Xerostomia has been described in cerebral palsy individuals submitted to drug therapy, surgery and botulinum toxin injections, as a form of drooling management.8
* Corresponding author at: Cieˆncias Biolo´gicas e da Sau´de – CBS, Universidade Cruzeiro do Sul, Rua Aure´lia, 1821, apto121 – Vila Romana, Sa˜o Paulo, SP, CEP 05046-001, Brazil. Tel.: +55 11 78727095. E-mail address: [email protected] (M.F. Leite). 0003–9969/$ – see front matter # 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.archoralbio.2010.07.016
856
archives of oral biology 55 (2010) 855–860
The organic and inorganic composition of saliva has several important biological functions. The use of saliva, crevicular fluid and mucosal transudates for drug monitoring and to detect several oral and systemic diseases has been described, particularly in autoimmune disorders, cardiovascular diseases, endocrinology, infectious disease, nephrology, oncology and psychiatry.9,10 The secretion of saliva and electrolytes is stimulated by the action of neurotransmitters of the autonomic nervous system. The concentration of salivary electrolytes depends on the action of co-transporters and membrane regulatory proteins, such as aquaporins,11,12 and on the action of the salivary duct system. The increase in intracellular concentration of ions Cl , due to the action of Na+–K+–2Cl and Cl – HCO3 channels, is responsible for an osmotic gradient that facilitates the secretion of potassium, sodium, bicarbonate and others salivary electrolytes.13 Saliva composition can vary according to salivary flow rate, the type of gland and hydration status.14 Water is the medium of circulatory function, biochemical reactions, metabolism, and substrate transport across cellular membranes, temperature regulation and numerous other physiological processes.15 Fluid-electrolyte turnover and whole-body water balance change constantly. Several methods have been used to measure the state of hydration in the body, including plasma osmolality, bioelectrical impedance spectroscopy, body mass change, urine tests and others.15 Osmolality is the concentration of a solution expressed in milliosmoles of solute particles per kilogram of water. It has been suggested that decreased levels of hydration (dehydration) may cause diminished salivary output.16 Some studies have shown that the salivary flow rate, total protein concentration and osmolality of saliva may be potential noninvasive markers of whole-body hydration status during intentionally progressive acute dehydration in healthy subjects.17 Moreover, theses parameters are strongly correlated with plasma osmolality (a widely accepted hydratation index), demonstrating that changes in saliva components reflect changes in hydratation status during hypertonichypovolemia.17 It has been clearly shown that a reduced salivary flow rate occurs in CP individuals.4–7 Reduced salivary flow rate could be either hyperosmolar or isosmolar. The measurement of osmolality, protein concentration and electrolytes could provide information regarding the real condition of salivary osmolality. To the best of our knowledge, no report regarding salivary osmolality status in individuals with CP has been published. The aim of this study was to measure the salivary flow rate, osmolality, electrolytes and total protein concentration in cerebral palsy individuals.
2.
Material and methods
2.1.
Subjects
This project was reviewed by the Human Research Ethics Committee and approved by the Federal University of Sao Paulo Institutional Review Board (IRB) and granted under protocol approval 1034/08. After being informed of the aim
of the investigation, written informed consent for participation and publication was obtained from the adult responsible for each child/individual who agreed to participate in the study. A group of 38 noninstitutionalized male and female children (aged 7–14 years-old) with CP, attending the Special Children’s Elementary School of Lar Escola Sao Francisco, Rehabilitation Center/Unifesp, were enrolled in this study. The inclusion criterion was a medical diagnosis of CP and the exclusion criteria were the use of any drugs that interfere with saliva secretion (anticholinergic and neuroleptic drugs, benzodiazepines) for at least 72 h prior to examination, a history of head and neck radiation and surgical procedures to reduce drooling. Patient medical records were reviewed for clinical data concerning the type of movement disorder (spasticity and dyskinesia). The control group consisted of 22 nondisabled children (aged 6–14 years-old) and the primary inclusion criterion was absence of neurological damage. None of the participants reported any complaints suggestive of salivary-gland dysfunction or were using any medication that could affect salivary secretion.
2.2.
Saliva collection
At least 2 h after the previous meal, unstimulated whole saliva was collected between 8 and 10 a.m., to minimise the circadian rhythm effects, using slight suction through a soft plastic catheter. Saliva produced in the first 10 s was discarded and the subsequent saliva was collected for exactly 5 min in a graduated cylinder to calculate the initial flow rate (mL/min). During the collection period, all individuals remained comfortably seated in a ventilated and illuminated schoolroom. If a child did not permit saliva collection, he or she was excluded. Soon after collection, saliva was frozen in dry ice, transported to the laboratory and stored at 80 8C until analysis.
2.3.
Measurement of salivary parameters
Salivary osmolality was measured using a freezing point depression Osmometer (Model Wide-Range Osmometer 3W2, Advanced Instruments, Massachusetts, USA). The sodium, potassium and chloride concentrations were measured using the respective Ion Selective Electrodes (AU400 Chemistry Analyzer, Olympus, Shizuoka-zen, Japan). Salivary total protein concentration was estimated by the Bradford method18 using bovine serum albumin as a standard.
2.4.
Statistical analysis
The data are presented as mean standard deviation (SD). Biochemical parameters of the groups studied were compared by Analysis of Variance and the Tukey test. The level of significance adopted was 5% ( p < 0.05).
3.
Results
Figs. 1–6 show the salivary parameters of healthy and CP individuals according to the neuromotor abnormality type
archives of oral biology 55 (2010) 855–860
[(Fig._1)TD$IG]
857
Fig. 1 – Salivary flow rate (mL/min) of healthy (control) (n = 22), total cerebral palsy (total PC) (n = 38), spastic (n = 29), and dyskinectic (n = 9) individuals. The data are expressed as mean W SD (columns and lines, respectively). Statistical differences compared to the control group are represented by asterisks (*) ( p < 0.05).
[(Fig._2)TD$IG]
Fig. 2 – Total protein concentration (mL/min) of healthy (control) (n = 22), total cerebral palsy (total PC) (n = 38), spastic (n = 29), and dyskinectic (n = 9) individuals. The data are expressed as mean W SD (columns and lines, respectively). Statistical differences compared to the control group are represented by asterisks (*) ( p < 0.05).
(total CP, spastic, and dyskinectic). The salivary parameters studied were osmolality, sodium, potassium, chloride and total protein concentrations and salivary flow rate. The results were expressed as mean SD. CP individuals with neuromotor abnormality type (total, spastic and dyskinectic) presented an increase in salivary osmolality (25%, 25% and 28%, respectively) (Fig. 3), total protein concentrations (14%, 14% and 19%, respectively) (Fig. 2), potassium (25%, 26% and 28%, respectively) (Fig. 5) and chloride (17% and 18% in the total CP and spastic, respectively) (Fig. 6) concentrations compared to the control group ( p < 0.05). Moreover, a reduction in salivary flow rate of total CP (29%) and spastic individuals (32%) was verified compared to the control group ( p < 0.05) (Fig. 1). No changes were observed in the sodium concentrations of the groups studied (Fig. 4).
4.
Discussion
Cerebral palsy is a disease which affects the motor functions of the individual and presents a series of oral manifestations, both clinical and biochemical. This study shows for the first time that individuals with CP present salivary changes characterised by an increase in osmolality, total protein, potassium and chloride concentrations. Moreover, the reduction in salivary flow rate in CP individuals observed in this study is in agreement with previous reports4–7 and supports the idea of hyperconcentrated saliva, which is more common in dehydrated status. Widely accepted and frequently used markers of hydration status include hematological and urinary parameters; however, the use of salivary parameters as indication of water loss have also been proposed.17,19 These studies showed that the
858
archives of oral biology 55 (2010) 855–860
[(Fig._3)TD$IG]
Fig. 3 – Salivary osmolality (mOsm/Kg H2O) of healthy (control) (n = 22), total cerebral palsy (total PC) (n = 38), spastic (n = 29), and dyskinectic (n = 9) individuals. The data are expressed as mean W SD (columns and lines, respectively). Statistical differences compared to the control group are represented by asterisks (*) ( p < 0.05).
[(Fig._4)TD$IG]
Fig. 4 – Sodium concentration (mEq/L) of healthy (control) (n = 22), total cerebral palsy (total PC) (n = 38), spastic (n = 29), and dyskinectic (n = 9) individuals. The data are expressed as mean W SD (columns and lines, respectively).
[(Fig._5)TD$IG]
Fig. 5 – Potassium concentration (mEq/L) of healthy (control) (n = 22), total cerebral palsy (total PC) (n = 38), spastic (n = 29), and dyskinectic (n = 9) individuals. The data are expressed as mean W SD (columns and lines, respectively). Statistical differences compared to the control group are represented by asterisks (*) ( p < 0.05).
archives of oral biology 55 (2010) 855–860
[(Fig._6)TD$IG]
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Fig. 6 – Chloride concentration (mEq/L) of healthy (control) (n = 22), total cerebral palsy (total PC) (n = 38), spastic (n = 29), and dyskinectic (n = 9) individuals. The data are expressed as mean W SD (columns and lines, respectively). Statistical differences compared to the control group are represented by asterisks (*) ( p < 0.05).
reduction in salivary flow rate and increase in osmolality and total protein concentration in the saliva of individuals subjected to procedures that lead to loss of water are correlated with other parameters of body dehydration. The individuals with CP who participated in this study presented similar results to individuals subjected to conditions of dehydration. The individuals of this study presented an increase in the concentration of electrolytes, i.e. potassium and chloride. These changes corroborate a previous study4 that suggested compromise of the duct system of salivary glands in individuals with CP. While passing through the duct system, the primary saliva secreted in the acini of the salivary glands undergoes reabsorption of sodium and chloride and excretion of potassium and bicarbonate.13 The submandibular gland undergoes changes in its secretory capacity, largely paracellular, according to the degree of osmolality of the medium.11 These changes are mediated by an osmosensor system in the basal membrane of the salivary gland.11 The hypohydration status probably causes an osmotic imbalance that alters the functions performed by the ducts of the salivary glands. Analysis of the results of this study suggests that individuals with CP present reduced salivary flow and changes in saliva composition due to lower water intake. The development of hypohydration in these individuals is due to the fact that they are mainly dependent on the initiative of being offering liquids by their caregivers. In addition, they do not complain of thirst and can hardly drink water by themselves. Moreover, CP compromises the ability of feeding skills,20 making it difficult to swallow all the water that is offered. Thus, those involved in the health care of individuals with CP should consider alternatives that would improve the hydration status of those under their care. The submandibular glands are the major contributors to resting mucous and serous saliva (unstimulated), while the parotid glands contribute to stimulated serous saliva.21 While the submandibular has a low oxygen demand, the parotid glands present high aerobic metabolism22 and therefore, are more prone to oxidative damage caused by reactive oxygen species (ROS). Water stress increases the formation of ROS resulting in lipid peroxidation, uncontrolled oxidation of thiol groups, denaturation of proteins and nucleic acid damage with severe consequences for overall metabolism. Other injuries
caused by hypohydration include reductions in cytoplasmatic and intracellular transport, alterations in cytoplasmic pH and cell membranes become more susceptible to attack by ROS.23 Unstimulated whole saliva was collected by slight suction without any difficulty.24,25 Previous studies reported alterations in salivary flow rate and organic and inorganic parameters of whole saliva in individuals with CP.4–6 The alterations observed in salivary total protein and electrolyte concentrations of individuals with CP could be caused by impaired functioning of the salivary glands, especially the submandibular glands. Thus, the results of this study lead to the conclusion that the diminished salivary flow rate and increased osmolality, total protein and electrolyte concentrations of saliva from individuals with cerebral palsy could be associated with hypohydration status. Moreover, hypohydration could be involved in possible oxidative damage in the salivary glands of individuals with cerebral palsy, further affecting the secretion of saliva.
Acknowledgment This study was supported by the Fundac¸a˜o de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) under protocol number 08/00960-6. Funding: This study was supported by the Fundac¸a˜o de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) under protocol number 08/00960-6, without any influence. Competing interests: There is nothing to declare. Ethical approval: This project was reviewed by the Human Research Ethics Committee and approved by the Federal University of Sao Paulo Institutional Review Board (IRB) and granted under protocol approval 1034/08.
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