Lipid peroxidation marker in saliva of Type 2 Diabetes Mellitus with oral manifestations – A Clinical and biochemical study

Original article: Lipid peroxidation marker in saliva of Type 2 Diabetes Mellitus with oral manifestations – A Clinical and biochemical study. Madi M1, Babu SG2, Kumari S3, Shetty SR4, Bhat S5, Castelino 6 Abstract: Background: Type 2 Diabetes progresses gradually and in a stepwise order. It commences with insulin resistance and progresses slowly with time until the body fails to maintain glucose homeostasis. These alterations are accompanied with changes in lipid peroxidation. The determination of the oxidative stress requires sometimes invasive techniques. Exploring saliva for oxidative stress has great clinical interestObjective: The present study was undertaken to estimate, compare and correlate the levels of malondialdehyde (MDA) in the serum and saliva of patients with type 2 diabetes mellitus with oral manifestations and healthy controls. Materials and Methods: Serum and salivary Malondialdehyde levels were estimated in 45 healthy subjects (Group I) and 45 patients with Type 2 Diabetes with oral manifestations (Group II). Estimation of Random blood sugar levels were done by GOD-PAP methodology. The data obtained from the present study was analyzed using SPSS software. Independent T test was used to compare the levels in the study and control group. Pearson’s correlation coefficient was used to correlate the changes in serum and saliva. P< 0.05 was considered significant. Results:The mean serum Malondialdehyde levels in Group I was 0.958 μM/l, while the mean serum Malondialdehyde levels of Group II was 2.828 μM/l. The mean salivary Malondialdehyde levels in Group I was 0.217 μM/l, while the mean salivary Malondialdehyde levels of Group II was 0.688μM/l. The mean serum and salivary Malondialdehyde levels were significantly increased in subjects with Type 2 Diabetes with oral manifestations in comparison to the healthy subjects. Fair positive correlation was observed between serum and salivary Malondialdehyde levels in Group I (r = 0.341) and very good positive correlation was observed between serum and salivary Malondialdehyde levels in Group II (r = 0.613). Conclusion: Serum and salivary Malondialdehyde was significantly higher in subjects with Type 2 Diabetes Mellitus with oral manifestations when compared to healthy controls. The increase in serum and salivary levels of MDA, shows that free radicals are formed disproportionately in diabetes mellitus by glucose degradation, non-enzymatic glycation of proteins, and the subsequent oxidative degradation, which may play an important role in the development of complications in diabetic patients. Fair positive correlation was found between serum and salivary Malondialdehyde in healthy subjects and very good positive correlation was observed between serum and salivary Malondialdehyde in subjects with Type 2 Diabetes Mellitus with oral manifestations. This study highlights that type 2 diabetic patients undergo abnormally high levels of oxidative stress. Hence exploring saliva for oxidative stress is of great importance. Thus saliva could be used as a reliable, non-invasive tool in the assessment of oxidative status.


Introduction:
Type 2 diabetes mellitus is a familiar endocrine and metabolic condition.It has touched epidemic magnitudes globally and symbolizes a grave community health concern.It is projected that it will be affecting roughly three hundred sixty six million people by 2030 1,2 .This rampant disorder is increasing at anextra ordinary rate in youngsters, with the major Bangladesh Journal of Medical Science Vol. 17 No.04 October '18.Page : 644-651  DOI: http://dx.doi.org/10.3329/bjms.v17i4.38330escalation in developing nations 1 .Diabetes mellitus is categorized by hyperglycemia.
Biochemically there are variations in glucose levels and lipid peroxidation.Lipid peroxidation is a process connected to free radicals.It is an uninhibited, self-augmenting process that leads to disruption of membranes, lipids and other cell constituents 3 .The occurrence of lipid peroxidation induced by free radicals cause considerable alterations in the cell membrane.
The lipid peroxidation of the membranes is induced by the reactive oxygen species (ROS).The destructiveness of the fatty acids peroxides that have been produced are the prime cause of failure of cellular functions.The most comprehensively used assay for lipid peroxidation is the measuring malondialdehyde (MDA).Therefore, the lipid peroxide in the blood offers evidence for the prognosis of diabetes.
The assessment of the oxida tive stress comprises procedures like blood sample collection that are quite invasive in nature.Whole saliva is a dynamic fluid which contains enormously complex constituents.Variable measures of blood and serum products are existing in whole saliva.Discovering saliva for oxidative stress has abundant clinical importance 4 .
Diabetes can be suspected on the basis of a multitude of systemic and oral signs and symptoms, including gingivitis and periodontitis, persistent oral fungal infections and compromised wound healingcapacity 5 .
Oral health is a mirror image of patients' general health.The capacity of saliva to be utilized to observe a patient's status of health and illness is a exceeding lydesired objective for promotion of health and research in the field of health sciences 6 .
Thus the present study is undertaken to estimate and compare the levels of malondialdehyde (MDA) in the serum and saliva of patients with type 2 diabetes mellitus with oral manifestations and healthy controls and to correlate the changes in the saliva and serum.

Materials and methods:
The present study was conducted on subjects who reported to Department of Oral Medicine and Radiology at a dental college in South India during 2013-2016.After obtaining the Institutional ethical clearance, the nature of the study and the purpose were explained to the participants in detail, and informed written consent was attained from the participants who were included in the study.A detailed case history of each subject was recorded with thorough examination of the oral cavity.Pocket depth was measured in millimeters using the probe at 4 sites per tooth.Clinical attachment loss was calculated using Williams graduated periodontal probe.Patients with periodontal disease having clinical loss of attachment more than or equal to 4mm were considered for study group (Group II).Patients should have maintained good oral hygiene with gingival index score of less than 2.0 for control group (Group I).The oral cavity of the subjects was also examined for any other oral manifestations of diabetes as mentioned in the inclusion criteria for Group II.

Method of collection of data: Sample collection:
Informed consent was taken from the patients included in the study.Ethical clearance was obtained from the Institutional Ethical Committee (Ethical Certificate Number: ABSM/EC/41/2012).

Saliva collection:
Samples of saliva were collected from participants two hours after intake of food using spit technique.The patient was made to sit on the dental chair with head slanting forward and asked not to speak or swallow saliva.Then the patient was asked to spit into a sterile container every minute for five to eight minutes.Salivary sample represents whole mouth fluid.The collected sample was centrifuged at 3000 rpm for ten minutes and the supernatant is collected and stored at -20 0 C. Blood collection: 5ml of venous blood was collected from the antecubital vein with syringe and placed in vials.Serum was then extracted and stored at temperature of -20 0 C in glass vials. 7: Random blood glucose level was determined by using glucose oxidase-peroxidase (GOD-POD) method.In this method the preliminary enzymatic oxidation of glucose is caused by the enzyme glucose oxidase (GOD).The calorimetric indicator is Quinone.It is produced by hydrogen peroxide from 4 aminoantipyrine and phenol because of the catalytic action of peroxidase (POD) (Trinder's reaction).3 test tubes each labelled as 'Blank', 'Standard' and 'Test' were taken.Then a pipette was used to transfer 1000 μl of reagent solution to each of these test tubes.10 μl of standard was added to the test tube marked as 'Standard'.This was followed by addition of 10 μl of test sample to the 'Test' test tube.The sample was mixed and incubated for 10 minutes at 37 0 C. Within 60 minutes absorbance was measured at 505 nm against reagent blank using Roche automated clinical chemistry analyser.

Treatment:
To the diluted sample one ml of TCA-TBA-HCl reagent was added.The samples were kept in boiling water bath for a period of fifteen minutes.The reaction mixture was cooled and centrifuged.The supernatant was taken and the optical density of the pink colour formed was read at 535nm.The concentration of malondialdehyde in the sample was got by plotting the obtained absorbance against the standard graph.The optical density of the pink colour formed was directly proportional to the concentration of malondialdehyde in the given sample.

Calculation:
The optical densities of the test samples were calculated by the plotting against the standard graph and multiplied by the respective dilution factors and the final concentration was expressed as µM/l.

Method of analysis
The data obtained from the present study was analysed using SPSS version 17.0 software.Independent t test was used to compare the levels in the study and control group.Pearson's correlation coefficient was used to correlate the changes in serum and saliva.p< 0.05 was considered as significant.

Results: Demographic data analysis of the groups: Demographic data analysis of Control Group (Group I):
The mean age in this group was 40.58

Analysis of mean serum and salivary Malondialdehyde levels in Controls and Type 2 Diabetics with oral manifestations: Mean Serum Malondialdehyde levels:
The mean serum Malondialdehyde levels in Group I was 0.958 µM/l, while the mean serum Malondialdehyde levels of Group II was 2.828 µM/ l[Table 3].

Mean Salivary Malondialdehyde levels:
The mean salivary Malondialdehyde levels in Group I was 0.217 µM/l, while the mean salivary Malondialdehyde levels of Group II was 0.688 µM/l [Table 3].

Analysis of Statistical Significance:
Serum Malondialdehyde levels: When serum levels of Malondialdehyde were compared between Group I (0.958 µM/l) and Group II (2.828 µM/l), the difference was statistically highly significant (p<0.001)[Table 3].

Correlation of Serum Malondialdehyde with Salivary Malondialdehyde levels among the two groups:
Group 1:Fair positive correlation was observed between serum and salivary Malondialdehyde levels.(r = 0.341) [Table 4 Graph 1].Group 2:Very good positive correlation was observed between serum and salivary Malondialdehyde levels.
(r = 0.613) [Table 4 Graph 1].It is projected that the incidence of DM in adult population will see a rise in the next two decades and considerable increase will be seen in the countries that are still developing.Moreover, it is predicted thatthe bulk of these patients would beaged between 45 and 64 years 10 .In our study the mean age of the patient in the study group consisting of Type 2 diabetics with oral manifestations was 47.91 years.Diabetes mellitus can have variable effects on the tissues in the oral cavity.Patients with poor control in their glucose levels are for the most part susceptible to severe persistent and recurrent bacterial and fungal infections 11 .Persistent poor glycemic control has been connected to the occurrence and advancement of diabetes as well as its associated complications, including gingivitis, periodontitis and alveolar bone loss.Patients with diabetes mellitus are susceptible to oral sensory, periodontal and salivary disorders.This has the capacity to escalate the risk of fresh as well as recurrent dental caries.Hypofunction of saliva also risesthe oral candidal carriage in adults with diabetes mellitus.Occurrences of burning mouth syndrome, glossodynia, lichen planus, neurosensory dysesthesias, salivary and taste dysfunction and xerostomia are also quite common among diabetics 5 .
Free radicals plays huge role in the origin and difficulties of diabetes mellitus 12 .Scavenging of reactive oxygen species may be unsuccessful due to the variations and perturbations in the endogenous free radical defence mechanisms.The consequences of this is the oxidative damage injury to the tissues 9 .Malondialdehyde, as TBARS (Thio Barbituric Acid Reacting Substances), is often used to measure the prooxidant/antioxidant equilibrium in type 2 diabetic patients as they are stable as well as easily measurable lipid peroxidation products 13  Mellitus with oral manifestations (r = 0.613).Thus, the lipid peroxidation evaluated in saliva of diabetic patients may be valuable in evaluating the activity and severity of the disease.The findings of this study suggests the role of saliva as an adjunctive tool to monitor prognosis of diabetes mellitus.This study suggests that exploring saliva for oxidative stress may have boundless clinical importance.So examining the salivary content of peroxidation of lipids in patients with type 2 diabetes mellitus that accurately reflect the severity of the oxidative stress is worthy.

Conclusion:
In the present study, both serum and salivary Malondialdehyde was significantly higher in subjects with Type 2 diabetes mellitus with oral manifestations when compared to that of the healthy individuals.Moreover, there was a positive correlation between serum and salivary Malondialdehyde in both the healthy subjects as well as subjects with Type 2 diabetes mellitus with oral manifestaions.The results obtained by comparison of serum and salivary levels of oxidative stress marker shows that the prospects for saliva as a non-invasive medium in screening and in monitoring prognosis of diabetes mellitus is abundant.This implicates a possibility for using saliva as an adjunctive diagnostic tool along with serum in the future.
Further extensive studies are required with larger samples to establish the reliability of Malondialdehyde in saliva as a potential biomarker of oxidative stress in diabetes mellitus and to establish the role of lipid peroxidation in the pathogenesis of diabetes mellitus and its complications.
8 : Principle: MDA reacts with thiobarbituric acid (TBA) to give a pink colour.This was read at 535nm.Both serum and saliva samples were analysed.Sample volume: Serum: 100µl Saliva: 250µl Chemicals that were used: 1) Tri chloro acetic acid (TCA)-(CH3COOCl3) 2) 2-thiobarbituric acid (TBA)-(C4H4N2O2S) 3) Hydrochloric acid (HCl) 4) Malonaldehyde bis (dimethyl acetal)-(C7H16O4) Preparation of the reagent: TCA-TBA-HCI reagent: -0.25N HCI: 2.21ml of cone.HCI is made upto 100ml with distilled water.-15% TCA and 0.375% TBA -15g TCA and 0.375g of TBA is dissolved in 100ml of O.25N HCI the reaction mixture was warmed to dissolve the contents and stored at 4°C.MDA Standard (Stock-164flg/ml): -16.4l of the standard malonaldehyde solution was taken and made up to 100ml with distilled water.MDA Standard (Working-1.64flg/ml):-100µl of the stock was made up to 10ml with distilled water Estimation of malondialdehyde in sample: Sample preparation: -Serum-100µl serum was diluted to 500µl with distilled water.-Saliva-250µl of the saliva was diluted to 500µl with distilled water.

Table 4 : correlation of salivary and serum levels using pearsons correlation
Individualsexisting with type 2 DM are more vulnerable to short term and long term complications.The higher morbidity and mortality rate is seen in patients with type 2 DM because of the commonness of this type of DM, its insidious and deceptive beginning and late recognition 10 .
21e present study is in accordance to the study conducted by Mahadevan et al20where high levels of MDA were observed in diabetics as compared to controls validating the role of oxidation of free radicals in pathogenesis of diabetes mellitus.The results established that salivary MDA is the indicator of oxidative stress in subjects with diabetes mellitus.Thus, saliva being minimally invasive and easy to collect can be used to assess MDA levels of the patients with diabetes mellitus.As seen in the present study, Rajeshwari et al21reported the increase in the salivary MDA levels and stated that diabetes mellitus can cause dysfunction in the endothelium owing to increased oxidative stress.Thus it could be stated that the overwhelming reaction of the body towards oxidative stress which is reflected in the saliva could be used as a reliable marker.No studies have documented the correlation between serum and salivary Malondialdehyde in healthy subjects and subjects with Type 2 Diabetes mellitus with oral manifestations.The Pearsons correlation analysis revealed fair positive correlation between serum and salivary Malondialdehyde in healthy subjects (r = 0.341).Very good positive correlation was observed between serum and salivary Malondialdehyde in subjects with Type 2 Diabetes