Estimation of Serum Procalcitonin (ProCT/PCT) Levels in Periodontally Healthy Individuals and Chronic Periodontitis Patients with Type II Diabetes Mellitus - An Original Research.

Aim: The present study aimed to estimate the serum procalcitonin (PCT) levels in periodontally healthy individuals and chronic periodontitis patients with Type II diabetes mellitus (DM). Materials and
Methods: Forty-five male subjects aged 25-60 years were enrolled in the study and grouped as Group I (healthy), Group II (chronic periodontitis), and Group III (chronic periodontitis with Type II DM). Clinical parameters (dental plaque scores, bleeding scores, probing pocket depth, and loss of attachment) and glycemic parameters (random blood sugar and glycated hemoglobin levels) were recorded. Serum procalcitonin levels were analyzed using Raybio® Human Procalcitonin Enzyme-Linked Immunosorbent Assay kit using the sandwich technique. All the data obtained were tabulated and analyzed using SYSTAT 12 statistical software. Kruskal-Wallis test was applied to compare the mean scores between the three study groups, and Spearman's ρ correlation coefficient was used to find out the association.
Results: Serum procalcitonin levels were markedly increased in periodontitis group when compared to the healthy group. The mean serum levels of procalcitonin in Group I, Group II, and Group III were 22.52 pg/ml, 64.23 pg/ml, and 185.86 pg/ml, respectively. The variation in the procalcitonin levels was statistically significant at P < 0.001.
Conclusion: The expression of procalcitonin in serum was increased to eightfold in the periodontitis group with diabetes in comparison to the healthy group, which shows that periodontal disease can cause the release of procalcitonin. Copyright:

INTRODUCTION

Gingivitis and periodontitis are inflammatory diseases with chronic nature, primarily caused by microorganisms and bacteria present in dental plaque. While gingivitis is mild and reversible form, periodontitis is more severe and destructive form. Periodontitis causes bacterial-induced endotoxin release which evokes pro-inflammatory cascade leading to secretion of tumour necrosis factor alpha and interleukin -1 beta, causing connective tissue destruction and bone resorption. Redness, swelling, pus discharge, loose teeth, spacing between teeth, exposure of root surfaces, and tooth loss are the common clinical features of periodontal disease.[1]

Diabetes mellitus (DM) is a medical condition characterized by high blood sugar levels (hyperglycemia) resulting from deficiencies in insulin secretion, altered insulin action, or both. It has been identified as an important risk factor associated with periodontitis. Hyperglycemia induces the formation and accumulation of nonenzyme-induced advanced glycation end products (AGEs). Increased cellular oxidative stress and activation of NF-κB on monocytes result from the interaction of AGEs and their receptor (Receptor for Advanced Glycation end products (RAGE)). This induces an increase in the production of pro-inflammatory cytokines (TNF-, prostaglandin E2, and IL1), as well as hydrolases and collagenases, resulting in severe connective tissue breakdown.[23]

Procalcitonin belongs to the calcitonin class of peptides encoded by the CALC-1 gene on chromosome 11 and has a molecular weight of 14.5 kDa. Thyroid neuroendocrine C-cells give rise to majority of procalcitonin. In subjects with good health, procalcitonin levels are as low as 10 pg/ml−1 CALC-1 gene expression is significantly increased in all parenchymal tissue and differentiated cell types in the body after a microbial infection causing a significant increase in its levels up to thousandfold than normal levels (1,00,000 pg/ml−1).[4]

Since diabetes and periodontitis have a well-established two-way interaction, the present study was aimed to estimate the levels of procalcitonin in serum of periodontally healthy subjects and chronic periodontitis patients with Type II DM.

MATERIALS AND METHODS

Institutional Review Board approval was obtained before the commencement of the research (IHEC/0116/2016). The research was carried on at Rajah Muthiah Dental College and Hospital, Chidambaram, India. The research included 45 male patients aged 25–60 years old. The patients were categorized into three groups:

Group I – Subjects who are both systemically and periodontally healthy (probing pocket depth ≤3 mm, bleeding on probing (BOP) <10%)

Group II – Patients with chronic periodontitis without Type II DM (probing pocket depth ≥4 mm; attachment loss ≥3 mm, BOP ≥50%)

Group III – Patients with chronic periodontitis with moderate to poorly controlled Type II DM (probing pocket depth ≥4 mm; attachment loss ≥3 mm, BOP ≥50%).[5]

The research comprised participants with moderate to poorly managed diabetes (HbA1c evaluation), with a diabetes duration of at least 5 years with a satisfactory compliance. Patients suffering from an aggressive form of periodontitis, patients having any previous history of addictions or drugs, systemic causes other than diabetes, or periodontal therapy before 6 months of the research that could influence the study's outcome were excluded. Since this was carried as a pilot research, the sample size was restricted to 45 people.

Sample collection

The following periodontal clinical values were obtained after a complete medical history and clinical examination.

Using a periodontal probe, an examination was done in the areas surrounding each tooth at buccal (mesial, mid, and distal) and lingual sites. Plaque Index,[6] Ainamo's Gingival Bleeding Index,[7] probing pocket depth,[8] and loss of attachment were measured.

5 ml of venous blood was harvested by venipuncture, and the extracted serum (1 ml) obtained after centrifuging blood at 3000 rpm for 5 min was transferred to a plastic vial and kept at 4°C right away. According to the manufacturer's instructions, samples were analyzed using a commercially available Raybio® Human Procalcitonin Enzyme-Linked Immunosorbent Assay kit based on the quantitative sandwich enzyme immunoassay approach. At 450 nm, spectrophotometric measurements of color change were taken. The concentration of serum procalcitonin was measured in picograms per milliliter.

Statistical analysis

Using the Statistical Data Analysis and Scientific Visualization (SYSTAT) 12 software, all data were tabulated and evaluated. Kruskal–Wallis test was applied to compare the mean scores between the three study groups, and Spearman's ρ correlation coefficient was used to find out the association.

RESULTS

Serum procalcitonin levels were markedly increased in periodontitis group when compared to the healthy group. The mean serum levels of procalcitonin in Group I, Group II, and Group III were 22.52 pg/ml, 64.23 pg/ml, and 185.86 pg/ml, respectively. The variation in the procalcitonin levels was statistically significant at P < 0.001 [Table 1].

Table 1

Kruskal-Wallis test for age, plaque index, gingival bleeding index, pocket depth, clinical attachment loss, and serum procalcitonin levels - group wise

Parameter	Group I healthy		Group II CP		Group II CP+DM		Kruskal Kruskal CP+DMee	P
	Mean	SD	Mean	SD	Mean	SD
Age	33.8	8.20	45.63	4.20	45.88	3.98	9.228	<0.001
PI (mm)	0.31	0.07	1.22	0.20	2.07	0.50	20.165	<0.001
GBI (%)	4.26	0.74	63.36	1.96	90.89	6.03	20.50	<0.001
PPD (mm)	3.08	0.16	6.14	0.24	7.06	0.49	19.022	<0.001
CAL	-	-	6.64	0.40	7.41	0.29	9.62	0.002
Serum PCT	22.52	1.07	64.23	2.25	185.8	2.89	20.480	<0.001

PI: Plaque index (mm), GBI: Gingival bleeding index (%), PPD: Pocket depth (mm), CAL: Clinical attachment loss (mm), PCT: Procalcitonin (pg/ml), CP: Chronic periodontitis, DM: Diabetes mellitus, SD: Standard deviation

Healthy people (Group I) were 33 years old, whereas diseased people (Groups II and III) were 45 years old. This indicates that periodontitis and diabetes became more common as the individuals became older. Furthermore, Group II crude protein (CP) and Group III (CP with DM) showed increased plaque accumulation, bleeding from the gingiva, and higher probing pocket depth in comparison with the healthy (Group I) with a statistical difference at P < 0.001.

Furthermore, the periodontal clinical measurements (Plaque Index, Gingival Bleeding Index, probing pocket depth, and clinical attachment level) revealed a statistically significant positive correlation with serum procalcitonin levels P < 0.01 [Table 2].

Table 2

Spearman’s ρ correlation coefficient between periodontal clinical parameters, glycemic parameters, and serum Procalcitonin

Parameters	Serum PCT
PI	0.856**
GBI	0.867**
PPD	0.889**
CAL	0.941**
HbA1c	0.621**
RBS	0.735**

**P<0.01. PI: Plaque index, GBI: Gingival bleeding index, PPD: Pocket depth, CAL: Clinical attachment loss, RBS: Random blood sugar, HbA1c: Glycated hemoglobin, PCT: Procalcitonin

DISCUSSION

Periodontal disease has been classified as the 6th most common diabetic consequence[9] and it is the most common oral problem among Type II diabetics. Although the precise impact of diabetes on the periodontium is unknown, several interacting factors such as pathogenic subgingival microbiota, alteration in host defenses,[1011] modified Poly-Morpho Nuclear (PMN) cell function, and inflammatory dysregulation may lead to a greater prevalence and severity of periodontitis in diabetic patients.[12]

In 1998, a model was presented by Grossi and Genco[13] to explain the molecular link between diabetes and periodontal disease. They went on to say that in diabetics, the “infection-mediated” periodontal route, along with insulin resistance, amplifies the traditional connective tissue degradation process. As a result of this, changes in periodontal parameters were evident clinically in terms of increased plaque, gingival bleeding values, probing pocket depths, and attachment loss. Between diabetes-affected individuals and unaffected individuals, there was a substantial difference in mean plaque values in our study, which is similar to the study by Bridges et al. 1996[14] where diabetic males had a considerably higher plaque index than nondiabetic men.

BOP has a 50% predictive value for future periodontal disease, and the absence of BOP shows no disease progression. The Gingival Bleeding Index was greater in diabetes individuals in our study, which is comparable to Tellervo Ervasti's study in 1985.[15] Furthermore, gingival bleeding was also more common in diabetics with poor control than in those with excellent or moderate control. Diabetes-related vascular alterations in the gingiva might be the cause of this increased bleeding.

According to Taylor et al., 1998,[16] subjects with type II diabetes had a fourfold greater risk of periodontal destruction when compared to those without diabetes. Khader et al., 2006[17] in their study, found that diabetics had a marked increase in pocket depths, loss of attachment, along with an increased number of mobile, and missing teeth than nondiabetics, this is similar to the results of our study.

Procalcitonin is a promising marker and prognostic indicator which exhibits greater specificity in identifying patients with bacteremia, sepsis, bacterial superinfection, and systemic inflammation. A rapid rise in serum PCT is seen in active inflammatory conditions. Since periodontitis is a bacterially-induced infection, initiated by Gram-negative pathogenic bacteria which release endotoxins which can lead to upregulation of procalcitonin. According to RS Redman, 2015,[18] serum-PCT levels were substantially higher in individuals with moderate-to-severe periodontitis (P = 0.036) and severe periodontitis (P = 0.048) as compared to the no periodontitis/mild periodontitis group. Furthermore, serum PCT was also shown to be superior to C-reactive protein as a marker for large-scale bacterial infection, such as sepsis or pneumonia, in patients with osteoarthritis and atrophic arthritis, indicating that serum PCT was superior in measuring periodontitis in arthritis patients.

According to research done by C. W. Bassim, in 2008,[19] the serum-PCT level of the patients with periodontitis and diabetes was 78 pg/ml, whereas in controls, it was 48 pg/ml with P = 0.04. Furthermore, the effects of local chronic infection, such as periodontitis, and systemic inflammation may compound and disrupt glucose control. This is coinciding with the results of our study. The increase in procalcitonin can be justified by the increased periodontal destruction occurring in Type II diabetic patients which shows that the periodontal parameters and serum procalcitonin have a definite connection.

CONCLUSION

The expression of procalcitonin in serum was increased to eightfold in periodontitis patient with diabetes when compared to the healthy group, which shows that periodontal disease could have caused release of procalcitonin. The future directions of this study would be with a greater number of study samples with age- and gender-matched controls, along with bacterial bio load assessment and estimation of procalcitonin following periodontal therapy.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.