160 12 - 15 years old school children who had smoker in their family were selected and divided into two equal cot+ (case) and cot¯ (control) groups. The selection criterion was cotinine kit. If the concentration of cotinine was ≥ 0.05 mg/mL they were categorized as cot+ and if less than that cot¯. The subjects were clinically healthy and had not received any antibiotics for the last 2 months and had no active caries.
3.1. Procedure
Parents of all participants signed written consent forms and helped the investigators to fill out the questionnaire. This study was conducted according to ethical code of university ethics committee and the proposal was approved by dental material of research center.
Subjects were matched according to age, gender and Loe and Silness plaque index. PPD was measured in all subjects with a Williams periodontal probe in four sites of the teeth which are mesiobuccal, distobuccal, buccal and lingual, and is determined by average of these sites. PPD is measured from the depth of pocket to the gingival margin. Before collecting the samples participants had to rinse their mouth with water for 1 minute to remove any existing debris or blood and were asked not to eat anything at least for 2 hours.
Two millimeter non-stimulated saliva was collected in test tube with spitting method 1 - 2 times every minute for 10 minutes. While collecting saliva the person was in sitting, comfortable position. After obtaining saliva the test tubes were covered by sealed parafilm and sent to biochemistry lab as soon as possible. Continin level of samples was investigated with ELISA method. Concentration of AST enzyme was measured by Pars Azmoon AST kit and Jenway GSO6 UV/Vis Spectrophotometer. Lactoferrin was measured by ELISA technique in biochemistry lab of Babol University of Medical Sciences.
3.2. Laboratory Steps
3.2.1. Lactoferrin Kit Lab Method
Lactoferrin EIA is an enzyme related immunologic method. The lactoferrin of samples is obtained through monoclonal antibody (Ab) that is placed on micro plates. Monoclonal Ab specific for lactoferrin attached to bioten is added to the samples. Lactoferrin is attached to antibodies and the sandwich is formed. Then, Streptavidin peroxidase solution is added. Streptavidin has tendency to bioten and with adding Ophenylenediamine the dye is formed. The intensity of this dye within the wavelength of 450 nm is in direct relation with lactoferrin.
3.2.2. AST Kit Lab Method
The basis of the test is that L-Aspartate reacts with 2-oxoglutarate by help of AST enzyme. In the next step oxoglutrate reacts with NADH by help of maleate dehydrogenase and maleate and NAD are produced. Different absorption of NADH in the wavelength of 340 nm is an indicator of AST activity.
3.2.2.1. Preparing Solution
This test can be done with one or two solutions. In this study single solution was chosen.
Indicator No I: LDH (Lactatedehydrogenase), MDH (Maleat dehydrogenase) L-Aspartate, TRIS Buffer (pH = 7.8).
Indicator No II: NAPH, 2-oxglutarate.
No I and II solutions were ready for use. For performing a single solution test, the solution No I and II were mixed with a 4 to 1 ratio (20 mL solution No I and 5 mL solution No II). 100 micro liter sample saliva and 1000 micro liter from the mixed sample were mixed. Light absorption was measured after 1 minute. The exact difference between light absorptions after 1, 2, and 3 minutes was measured. The total of these three values was divided into 3 and multiplied by 1985.
3.3. Statistical Analysis Methods
The data was analyzed by SPSS 18 and t-test and Mann-Whitney U test.
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