To analyze and Compare C-reactive Protein levels in GCF and Serum in patients with Coronary Heart Disease

INTRODUCTION

Periodontitis is one of the most prevalent and multifactorial disease in which microorganisms plays the major role. On an average estimate, periodontitis seems to affect 70% of the world population. Periodontal disease is interplay between inflammation, immunity and mechanism of the host to microbial invasion.

It is well known that, periodontal infection may affect the onset or the progression of CHD through certain common mechanisms. Periodontitis and CHD both have complex etiological factors, combining genetic and environmental influences in addition to smoking, the disease share many risk factors and have distinct similarity in basic pathogenic mechanism. One theory is that oral bacteria can affect the heart when they enter the blood stream, attaching to the fatty plaques in the coronary arteries (heart blood vessel) and contributing to clot formation¹.

Destefano et al² , demonstrated in patient a positive relation between and increase incidence of CHD. Since dental caries and periodontal disease are chronic infections that are often asymptomatic, they could be the source of increase levels of C-reactive protein, and acute-phase protein that have suggested as a predictor of myocardial infarction and stroke³. Evidence by Liuzzo et al⁴, suggest that C-reactive protein is elevated in patients with unstable angina and also at the time of hospital admission in all patients with Myocardial Infarction.

Limited studies are available to show presence of C-reactive protein in gingival crevicular fluid. Thus this study was attempted to correlate C- reactive protein in serum and gingival crevicular fluid samples of CHD patients with periodontitis. This study also was aimed to find out whether C-reactive protein in gingival crevicular fluid can be used as a marker in detection, high risk patients for CHD.

In a recent study by Beckman et al,⁵ an increase in C-reactive protein levels was associated with significantly increased risk for a cardiovascular event, and ray et al⁶ reported that reducing the C-reactive protein level can lead to decrease in mortality and morbidity.

MATERIALS AND METHODS

The study consisted of 60 patients between the age group of 20-60 years which included both sexes.The patients were categorized into 3 groups.

Group A: Consists of 20 patients having periodontitis with CHD.

Group B: Consists of 20 patients with only periodontitis and no other systemic disease.

Group C: Consists of 20 patients with healthy periodontium which served as controls.

Inclusion criteria:

Patient with adult periodontitis with pocket depth >5 mm.All patient selected should not have undergone any periodontal treatment preceding the study. Controls were those with clinical probing depth < 3 mm and with no other systemic disease.

Exclusion criteria:

Patient on antibiotic or with any acute-infections. Pregnant and lactating mothers. It was made clear that all potential subjects that participation was voluntary and written consent was obtained from those who agreed to participate. All the selected patients were subjected to OHI-S index (Green and Vermillion 1964) and CPITN Index (Ainamo and associate 1982) and their scores were recorded in a prepared proforma.

Method of collection of GCF:

The sites selected for GCF sampling (4-13 teeth) were isolated with cotton rolls and are dried for 2 seconds. Supragingival plaque obstructing access to the entrance of the crevice was carefully removed using a sterile periodontal probe.

Glass micro-pipettes (0.5 µl volume) were placed at the opening of the gingival crevice and fluid was collected for a period of 15-20 minutes. It was then transferred to a plastic vial containing 30 µ lof Bovine serum albumin, and stored at - 70°C until assay.

Method of collection of blood:

Blood samples were collected from anticubital fossa with the help of a disposable 22 gauge needle with syringe. It was then transferred to a test tube, allowed to clot, and it was centrifuged for 10 mints to get serum. The serum was stored at -70°C until assayed.

Estimation of CRP level:

The GCF and serum samples were transported to Laboratory, for estimation of CRP level by a method based on an "Enzymatic heterogeneous sandwich immunoassay format' technique.

Technique:

Assay Time and Temperature: 7.5 mints at 37°C.

Enzymatic heterogenous sandwich immunoassay format (Vitros Chemistry, Johnson and Johnson Company).

This consists of vitros CRP slide which is dry, multilayered, analytical element coated on a polyester support.

In this format a derivative of phosphorylcholine (PC) is covalently bound to polystyrene polymer beads and in the presence of calcium serves as a capture agent. Monoclonal anti-CRP antibody conjugated to horseradish peroxidase (HRP) serves as a signal generator.

An 11 µl drop of patient sample is deposited on the slide and is evenly distributed by the spreading layer to the underlying layers. CRP in the sample binds to PC-linked capture beads and anti-CRP antibody labeled with horseradish peroxides to form an insoluble sandwich complex. The subsequent addition of vitrosImmuno wash fluid to the slide removes unbound materials from the read area, while also providing the H₂0₂ required for the enzyme mediated oxidation of  leukocyte. The reflection density of the dye is measured at 670 nm 2.5 mints after the addition of Vitros Immuno-wash fluid. This reflection density is directly proportional to the concentration of CRP in the sample. To determine if an adequate wash has occurred, the wash detection dye is read at 540 nm during the final 2.5 mint incubation period.

Reaction Sequence:

PC = phosphorylcholine beads

Ab.HRP = anti - CRP monoclonal antibody labeled with horseradish peroxidase

RESULTS

The means obtained from the data of each group were subjected to student't' test.The results of the present study were compared and evaluated to find out the mean CRP levels in serum and GCF samples of all the 3 groups.( Table 1).

The mean OHI-S index for group A is 3.91 and for group B is 4.05 and the mean of the CPITN index for group A is 3.75 and group B is 3.95, which is not statistically significant (P>0.05) Table 3. The results of the study reveal that OHI-S and CPITN index were reported to show similar trends in case of group A and group B.Most of the patients in the study were reported to have poor Oral hygiene with score 2 & 3 and CPITN score of code 3 & 4, which requires oral hygiene instructions and complex periodontal treatment needs.

CRP levels in serum:

The results were evaluated for mean CRP levels in serum for all 3 groups with standard deviation. The data was subjected to statistical analysis using student 't' test.The result were compared between 3 groups showed a t value of (p <0.05) the inference was found to be statistically significant. (Table 2).

Both group A and group B when compared with the controls showed to be statistically significant.

CRP Levels in GCF:

The results were evaluated for the mean CRP levels in GCF. The data was subjected to statistical analysis using student 't' test.

The results for the CRP levels of activity were compared between 3 different groups.

Showed 't' value of (P < 0.05), the inference is that the difference in the CRP values between the 3 groups were significant. (Table 2).

The serum to GCF values was determined to indicate the severity and has a prognostic indicator of the 2 diseases studied, compared to the controls. The results obtained for GCF was similar between the group A and B but was significant when compared with the control GCF values.

The results of the serum values between group A and group B were positively co-relating with each other, but were significant when compared with the control serum values.

The serum to GCF were determined and found to indicate that serum CRP was 20 fold higher than GCF CRP values in group A.For group B the serum CRP values was 5 fold higher, than the GCF CRP values.

DISCUSSION

Periodontitis is a local inflammatory process mediating destruction of periodontal tissues triggered by bacterial insult. The disease is also characterized by systemic inflammatory host responses that may contribute to high risk for cardiovascular disease. G.R. Perrson reported the association between Cardio Vascular Disease (CVD) and periodontal disease, and found that pathologically altered periodontal pockets may allow virulent bacteria to enter the blood stream, adhere, colonize and invade surface cell barriers such as those of pocket epithelium or vascular endothelium. Periodontal pathogens with outer membrane lipopolysaccharide capsules can induce production of significant amounts of cytokines linked with pathogenesis in both CVD and Periodontal Disease⁷.

Chronic bacterial infections such as periodontitis are one of the established risk factors which contribute for moderate elevation of CRP levels⁸. Studies have shown that periodontal infections results in activation of acute phase reactants proteins (APR) and elevation of certain serum markers (Gore et al., 1998)⁹. In a very recent study, CRP proved to be the strongest and most significant predictor of the risk for future cardiovascular events among several plasma variables¹⁰.

It appears likely that CRP and perhaps other markers of APR will serve as important diagnostic tools to evaluate whether periodontitis has any systemic involvement, hence the present study was designed to find out possible correlation between periodontitis and CHD.

CRP estimation was selected because of its rapid and dramatic rise in blood level following trauma or in diseases producing inflammation. The rapid increase of CRP is serum following exposure to IL-1 (Gewurz et al., 1982)¹¹ which is a potent bone resorber is also found in GCF (Mergerhagen 1984), This made the search for elevated CRP levels in inflamed periodontal tissues of CHD patients.

In the present study participants patients were asked for history of any antibiotics and periodontal treatment or surgeries prior to the study, because it has been shown that serum levels of CRP was found to increase in response to surgical procedures and sequentially begins to decline gradually postoperatively (Fischer et al., 1976)¹².

Comparison of CRP values between group A and group B showed a statistically significant difference (P < 0.05). The high CRP Values in group A patients may be attributed to the underlying systemic disease CHD when compared to periodontitis patients. This is in aggrement with study of Beer et al (1982)¹³.The results of this study is further supported by the findings of Berk et al (1990)¹⁴.

The present study showed that in some individuals of periodontitis group the CRP level in serum was undetected. Hence a definite correlation could not be drawn between serum values and its relationship to periodontal disease. This is in contrast with study of Ebersole et al, where an increase levels has been found in serum of all patient with periodontitis¹⁵.

The serum CRP levels between CHD patients with periodontitis and controls and periodontitis with controls have shown statistically significant difference. But the values for CRP in serum was 0mg/dl, in controls which might depend upon the sensitivity of the technique, in which it cannot detect the trace amount of CRP in the serum.This is in contrast with study of Shine et al., (1981) where they found in normal healthy subjects CRP is present in trace plasma protein with median level of 0.8mg/liter and dramatically rise in response to bacterial infection, trauma, tissue necrosis and most forms of inflammation, and may exceed 300mg/liter within 48h of an acute event¹⁶. High levels may persist indefinitely in chronic inflammatory states. However, according to Berk et al, it was found that CRP is present in normal has 0-1 ng/ml.

Ridker et al (2002) in his study found that CRP levels >2mg/l seemed to reflect inflammation and an elevated risk for cardiovascular disease^17,18. But until recently, CRP values< 10mg/l were considered normal, while acute bacterial infections have been reported in 80% to 85% of patients with CRP values > 100mg/l.¹⁹

The GCF CRP levels in group A and group B patients showed positive correlation indicative of active periodontal disease, but in some it was undetected. This is in agreement with sibraa et al²⁰.

According to pearson et al, individuals with hsCRP levels less than 1.0, 1.0-3.0 and greater than 3.0mg/L portend low, intermediate and high risk respectively, for cardiovascular disease²¹. However a recent study on using high-sensitivity CRP also found that significant elevation in serum CRP but with GCF it did not differ from those of the control.²²

Another study by Pradeep A.R et al, that CRP in GCF and serum was found to increase proportionately with severity of periodontal disease and also correlating positively with clinical parameters.²³ A similar study conducted by Isaac suzartgomes –filho et al, inferred that chronic periodontitis may increase the inflammatory burden simultaneously raising the plasma CRP levels which may lead to higher risk for  developing CHD²⁴.

In summary, the CRP levels in control group found to be zero in the present study may be attributed to the sensitivity of the technique employed in which it is unable to detect the trace quantity of CRP in ng/ml. The technique employed in the present study is a biochemical method using enzymatic sandwich immunoassay format which can detect up to 0.7mg/dl of CRP in the given sample. It is conceivable therefore that further sensitive method which can detect ng/ml of CRP in serum and GCF can be undertaken for precise detection & correlate between CHD and periodontitis.

CONCLUSION

In the present study, CRP in serum of group A and group B demonstrated that there is an increase, hence it can be consider as an indicator of active periodontitis and may be a risk for developing CHD. However there is no possible correlation with respect to CRP in gingival crevicular fluid, hence it is recommended that more number of longitudinal studies in a large population is necessary to enlighten CRP as a risk for CHD. Furthermore it is suggested to use more sensitive technique to estimate CRP level in the gingival crevicular fluid.