Brazilian Journal of Oral Sciences Piracicaba Dental School - UNICAMP EISSN: 1677-3225 Vol. 10, Num. 2, 2011, pp. 93-97

Salivary characteristics of diabetic children

Gheena S.¹, Chandrasekhar T.², Pratibha Ramani³

¹MDS, Senior Lecturer, Department of Oral and Maxillofacial Pathology, Faculty of Dental Sciences, Sri Ramachandra University, Chennai,
India ²MDS, Professor and Head of Department, Department of Oral and Maxillofacial Pathology, Saveetha Dental College, Saveetha University, Chennai, India
³MDS, Professor, Department of Oral and Maxillofacial Pathology, Saveetha Dental College, Saveetha University, Chennai, India

Received for publication: July 15, 2010 Accepted: May 31, 2011

Correspondence to: Gheena.S Dept of Oral and Maxillofacial Pathology, Faculty of Dental Sciences, Sri Ramachandra University, Porur, Chennai, India Phone: +91 - 98840 33777 E-mail: gheena_ranjith@yahoo.co.in

Code Number: os11019

Abstract

Aim: The objectives of this study were to evaluate the levels of glucose, cholesterol, protein and albumin in saliva, and to correlate the levels of glucose of the saliva to oral health and blood glucose of diabetic and non-diabetic children.
Methods: 32 children with type 1 diabetes mellitus formed the study group (DC) and 32 non-diabetic children formed the control group (ND). The patients had their saliva collected and evaluated for glucose, cholesterol, total protein and albumin. Blood glucose analysis was also performed. The dental health status of the subjects was measured by DMFT index and def index. Independent Student's t-test was performed to compare metabolic status values in DC and ND groups. Correlation test was applied between blood glucose and salivary glucose (Spearman's correlation), and salivary glucose and DMFT/def (Spearman's test).
Results: A statistically significant difference was observed between DC and ND considering salivary glucose (p=0.000). Elevated levels of cholesterol were evident in DC in correlation with ND. Total protein and albumin had increased values in DC (nonsignificant p value). The dental health status was not statistically different.
Conclusions: Salivary parameters can act as adjuncts in assessing the overall metabolic status of the patient.

Keywords: saliva, diabetes, pediatric, glucose, cholesterol.

Diabetes mellitus (DM) comprises a group of common metabolic disorders thatshare the salient feature of hyperglycemia. Several distinct types of DM exist and arecaused by a complex interaction of genetics, environmental factors and lifestyle choices.The metabolic deregulation associated with DM causes secondary pathophysiological changes in multiple organ systems and imposes a tremendous burden on the individualwith diabetes and the health care system.

Although all forms of DM are characterized by hyperglycemia, the pathogenic mechanisms by which hyperglycemia arises differ widely. The two broad categories ofDM are designated as type1 (IDDM) and type 2 (NIDDM). The incidence of childhoodonset type 1 diabetes is increasing in many countries in the world, at least in the under15-year-old age group. There are strong indications of geographic differences in trendsbut the overall annual increase is estimated to be around 3%.Two international collaborative projects, the Diabetes Mondiale study (DiaMond)¹ and the Europe andDiabetes study (EURODIAB)² have been instrumental in monitoring trends in incidence.

By 2010, it is estimated that annually some 76,000 children aged less than 15years develop type 1 DM worldwide. Of the estimated 480,000 children with type1 DM, 24% come from the South-East Asian Region, but the European Region,where the most reliable and up-to-date estimates of incidence are available, comesa close second (23%)³.The incidence rate of type 1 DM in India is 4.2/100,000population per year.

A wide spectrum of oral manifestations of DM has beenreported ranging from xerostomia, taste impairment, sialosis,dental caries and periodontal disease to fungal infections,oral lichen planus and fissured tongue⁴. However, in children, there is no agreement in results that relate alterations ofsalivary chemical composition and oral health⁵.

An optimum metabolic control level correlates positivelywith the well being of the patient. Assessing the metaboliccontrol of the patient has traditionally been done throughtesting of glycosylated hemoglobin, fructosamine, andglycoalbumin along with other criteria such as Body MassIndex, microalbuminuria and dyslipidemia^6-7. Fructosamine reflects the glycemic control over the previous 1-2 weeks and has been correlated positively with serum albumin and serum total protein⁵. Saliva is a unique fluid and interest init as a diagnostic medium has advanced exponentially inthe last decade. Advances in technology have helped to movesaliva beyond evaluating oral health characteristics to whereit now may be used to measure essential features of overallhealth⁸.

Thus, the objectives of this study were to evaluate thelevels of glucose, cholesterol, protein and albumin in saliva,and to correlate the levels of glucose of the saliva to oralhealth and blood glucose of diabetic and non-diabetic children thereby analyzing whether saliva-based glucose testing can be an effective alternative to blood-based glucose testing in children.

Material and methods

Study population

The study group comprised 32 diabetic children (DC) aged between 5 and 15 years with an already establisheddiagnosis of type 1 DM. This group was selected from thepatients attending the outpatient diabetes department, Institute of child health and research centre, Egmore, Chennai.The control group comprised 32 non-diabetic children (ND)without any other pre-existing systemic diseases. The study was conducted in collaboration with the Institute of Child Health and Research Centre and College of Dental Surgery,Saveetha University, Chennai with the ethical committee approval [(ihep no.) MDS 34/SU 63/06]. Parental informed consent was obtained for all patients before they were examined and samples collected.

Sample collection was performed during the morning hours when the subjects are in the fasting mode. Additionally, blood glucose values determined from venous blood samplesdrawn at the time of collection of saliva (fasting blood sugar)were also taken into account.

Standard UCLA procedure was used to collect the saliva.The subjects were asked to refrain from eating, drinking orother oral hygiene procedures, for at least one hour prior tocollection. Drinking water was then given to the subjects, torinse their mouth. Five minutes after the oral rinse, unstimulated saliva was collected in 50 mL falcon tubes bythe method of spitting the saliva. The patient was asked to swallow the saliva present in the mouth and then to remain still without moving the tongue or swallowing the saliva forone minute. The patient spat the saliva every 60 s for a totalof 5 min into a falcon tube to which sodium fluoride⁹ has been added. The tubes were then placed on ice. About 1.5mL of saliva was collected from each subject. The samples were then centrifuged at 2,500 rpm for 5 min. Thecentrifugation resulted in a saliva sample which is free oflarge particulate debris and reduced viscosity, thereby allowing a more accurate and reproducible analysis.

Glucose was estimated by using a glucose kit (CRESTBIOSYSTEMS, A division of Coral Clinical Systems, Goa,India) based on glucose oxidase-peroxidase method. It was standardized for saliva with 0.384mg/dL - as the lower value.The absorbance of the standard and the test material (0.01mL/10 µL) were measured against the blank without dilution within 60 min at a wavelength of 505 nm (Hg 546 nm)/Green. Total glucose in milligram/deciliter (mg/dL) is thenderived as Absorbance of test (Abs.T)/ Absorbance of standard(Abs.S) X 100.

Cholesterol (CH) was estimated by using a commercially available cholesterol kit, which is based on cholesterol oxidase /PAP method. The absorbance of the standard andthe test material (0.01 mL/10µL) without dilution were measured against the blank within 60 min at a wavelengthof505nm (Hg 546nm)/ Green. Cholesterol in mg/dL = Abs.T/Abs. S X 200.

Total protein (TP) was estimated by using a commercially available test kit based on the biuret method. The absorbance of the standard and the test material (0.02 mL/20 µL) aremeasured against the blank without dilution within 60 minat a wavelength of 550 nm (Hg 546 nm)/ yellow  green.Total proteins in gram (g)/dL = Abs.T/Abs.S x8

Estimation of albumin (ALB) was done by the colorimetric method based on the BCG method (commercially available test kits). The absorbance of the standard and thetest material (0.01 mL/10 µL) are measured without dilution against the blank within 60 min at a wavelength of 630 nm(Hg623nm)/ Red. Albumin in g/dL= Abs.T/Abs.S x 4.

The dental health status of the subjects was measuredby the DMF-T index and the def index. The DMF indexpertains to the permanent teeth (D - Decayed, M- Missingand F - Filled) and the def index (d - decayed, e - extracted, f - filled) to the deciduous teeth

Statistical analysis

Independent Students't-test was performed to compare metabolic status values in DC and ND groups. Correlation test was applied between blood glucose and salivary glucose(Spearman's correlation), and salivary glucose and DMFT/def (Spearman's test).

The mean value of salivary glucose (SG) of DC is 10.4mg/dL and for ND is 17.6 mg/dL. The resulting p = 0.000was 99.9% significant. A statistically significant difference(p = 0.000) was found between DC and ND with regard to cholesterol values. No statistically significant difference wasfound between DC and ND with regard to albumin (p =0.22) and total protein (p = 0.47) (Table 1).

There was no significant correlation between salivary glucose (SG) and blood glucose (BG) within DC, but there was a significant correlation within ND (p=0.000) (Table 2). The correlation between the DMFT status and the salivary glucose values of the subjects yielded nonsignificant results.

Whole saliva is a mixture of the secretions produced bythe three pairs of large glands and the smaller glands of oralmucosa (labial, lingual, buccal and palatal). It may alsocontain fluid from the gingival pocket (gingival, or crevicularfluid)⁹. The serum constituents in saliva are derived from the local vasculature of the salivary glands as well as from gingival fluid. The increased salivary glucose evident in thewhole saliva can be due to several contributing factors or itcould be a simple reflection of the blood glucose levels sincesaliva is an ultra filtrate of plasma. The salivary analytes arederived from plasma generally by three mechanisms (passive diffusion, active transport and ultra filtration) and are thus found in saliva as mentioned by Miller¹⁰.

A chronic disease like DM, with its emphasis on patient involvement in the control of the disease, and its attendant Correlation between blood glucose and salivary glucose in diabetic children and non-diabetic children problems, place a huge burden on the pediatric patients. Our efforts should be directed at making it an easier burden to bear. If saliva could be used for diagnosis and monitoring,diabetic children would not require daily invasive tests, they can just collect their saliva in a sterilized collection tube,which could then be used in biosensors for the real-time, sensitive and specific detection of salivary diagnostic analytesto give an overall view of the child's diabetic status. Very little information is available on the potential of saliva indiabetic children in India.

The salivary glucose values of the case subjects in ourstudy were lower than the controls. This finding is similar tothose of previous studies^11-13. Reuterving et al.¹¹ determined whether salivary flow rate and salivary glucose concentration in patients with diabetes mellitus influence of severity ofdiabetes. Salivary glucose concentration was lower duringthe period of better metabolic control. Marchetti et al.¹² found that the salivary glucose secretion rate was significantly lower(p less than 0.02) in diabetic patients with diabetic autonomic neuropathy than normal patients. Kanji et al.¹³ measured the plasma and salivary concentrations of glucose and cortisolduring insulin induced hypoglycemic stress in healthyNigerians. Salivary glucose levels (fasting and after intravenous insulin) were unaffected by hypoglycemia anddid not correlate with plasma glucose at any time point.

We could not ascertain the reason for the low salivary glucose concentrations evident in DC in comparison to ND.One of the confounding variables may be that the cases inDC were under treatment as opposed to the cases in ND.More studies are needed on this facet.

The salivary concentration of Lipid soluble, unconjugatedsteroids such as cortisol, estriol, testosterone and progesterone,closely reflects the plasma concentration. The significantly high values of salivary cholesterol seen in the diabeticsubjects could be an indication of the dyslipidemia, a condition usually associated with diabetic subjects^14-15.

Dyslipidemia is a common feature of DM and needs tobe treated because of the potential complications especially atherosclerosis. Cholesterol being a lipid moiety passes intosaliva by passive diffusion and has the potential to closelyreflect blood concentrations.

The cholesterol of case subjects is statistically higherthan that for the control subjects. Karjailanen et al.¹⁶ suggested that salivary cholesterol may be regarded as atransudate from serum, as suggested by Slomiany et al. The positive correlation of serum and saliva cholesterol valuesfurther supported the concept that at least part of the salivarycholesterol originates from serum. They also concluded that,in healthy adults, salivary cholesterol concentrations reflect serum concentration to some extent and can be used to select individuals with high serum cholesterol levels. MedicalLaboratory Observer (2000) gives information about a salivabased cholesterol test that has demonstrated the potential tocorrelate a quantitative result with patient blood cholesterollevel.

Ben Aryeh et al.¹⁷ found no difference in salivary totalprotein, amylase, lactoferrin, or lysozyme among the threegroups (IDDM, NIDDM, Controls) examined. Streckfus etal.¹⁸ recorded that all diabetic groups in their studydemonstrated a significantly lower salivary total proteinconcentration when compared with controls, which is incontrast to our study values.

There is a lack of literature on total protein and albumin estimations from the saliva of diabetic subjects, and the available data do not show a consensus on the results. These varied results indicate, as suggested by Rantonen et al.¹⁹ that these proteins are subject to short-term variation. Improved ways of evaluation of these elements in saliva with sophisticated techniques or another way of assessing themetabolic control in saliva needs to be considered.

The diabetic group and the control group did not differwith regard to the dental status as opposed to the usualscenario where diabetic children are expected to have morecarious lesions. This could be attributed to the reduction in the frequency of sticky food intake and food with highglycemic index and better oral hygiene practices by thediabetic children. Swanljung et al.²⁰ found that if the patients' IDDM is well controlled, their salivary and caries data doesnot differ from that of healthy controls. Blanco et al.²¹ found no differences in the number of decayed, missing and filledteeth based on metabolic control, evolution time and existence of late complications of diabetes.

The lack of correlation between the salivary glucoseand blood glucose values in DC underscores the importance of more studies needed in this direction. Both saliva and blood samples were taken during morning hours when thecase subjects were in fasting mode. One of the confoundingvariables may be the fact that the cases in DC were undertreatment as opposed to the cases in ND.

The cholesterol in DC was significantly higher than thatof ND. Further research is needed, especially on the correlationof salivary and blood values of cholesterol, to have a better understanding of this relationship.

Total protein and albumin can be used to assess themetabolic status of diabetic subjects since both showed aslight increase as compared to controls. The dental cariesexperience correlation between the cases and controls was not significant and gives credence to the value of diet andoral hygiene measures in the subjects. Salivary parameterscan act as adjuncts in assessing the overall metabolic statusof the patient. Further studies should be performed to make these tests equivalent to a blood test.

The authors express their sincere gratitude to Dr. N.Malathi, Faculty of Dental Sciences, SRU; Director, Instituteof Child Health; Dr. Parivardhini, Diabetes OPD, Institute of Child Health; Mr. Porchelvan and Mr. Ravi Shanker, statisticians, Sri Ramachandra Medical College and Researchinstitute for their help and motivation.

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