Effects of initial periodontal therapy on leucine-rich alpha-2 glycoprotein levels in saliva from Japanese patients with chronic periodontitis

Abstract

Purpose: Examination of patients to detect periodontal disease is important for diagnosis and treatment planning, and accuracy of examination may be improved if salivary components can be applied for diagnosis. Leucine-rich α2 glycoprotein (LRG) is expressed in the serum of patients with inflammatory diseases, and salivary LRG may be applicable to the diagnosis of periodontal disease.

Methods: To evaluate the effect of initial periodontal therapy (IPT) on clinical periodontal parameters such as probing depth (PD), clinical attachment level (CAL), bleeding on probing (BOP), periodontal inflamed surface area (PISA), periodontal epithelial surface area (PESA), saliva samples were collected before and after IPT from 63 patients with chronic periodontitis. The amount of LRG protein in saliva was measured by enzyme-linked immunosorbent assay.

Results: Salivary LRG levels of 30 patients with Stage III, Grade B or C periodontitis (Severe group) were higher than the LRG levels of 33 patients with Stage Ⅰ or Ⅱ, Grade A periodontitis (Mild group). LRG levels in the Severe group significantly decreased after IPT. Positive correlations were found between salivary LRG levels and mean PD, CAL, BOP rate, PISA and PESA.

Conclusion: These results suggest that there is an association between salivary LRG levels and severity of periodontitis.

Introduction

To perform periodontal therapy appropriately, it is important to examine the symptoms of current periodontal disease accurately and make a diagnosis. Periodontal disease examination includes periodontal tissue and bacteriological examinations. The periodontal tissue examination evaluates destruction of the periodontium based on probing depth (PD), bleeding on probing (BOP), clinical attachment level (CAL) and the presence or absence of bone loss [1]. Gingival inflammation is evaluated using the gingival index (GI) [2] and BOP [3,4]. In addition, bacteriological tests such as quantitative polymerase chain reaction (qPCR) tests and serum antibody levels against periodontal pathogens are performed using subgingival plaque, saliva and serum [5,6]. The periodontal inflammatory surface area (PISA) is the surface area of the bleeding pocket epithelium in square millimeters and helps to quantify the amount of inflamed periodontal tissue and thus indicates the severity of periodontal disease [7]. Expression levels of interleukin-1β (IL-1β) were increased in the gingival crevicular fluid (GCF) from deep periodontal pocket (PD ≥5 mm) compared with shallow periodontal pocket (PD ≤3 mm), and salivary IL-1β levels are raised in the patients with chronic periodontitis, which are reduced after initial periodontal therapy (IPT) [8,9]. These results suggest a close relationship between IL-1β levels in GCF and saliva and severity of periodontitis and that IL-1β might be useful as a biomarkers for periodontal disease. Saliva is non-invasive and easy to collect, and could be extremely useful if applied to the diagnosis of periodontal disease.

Leucine-rich α2 glycoprotein (LRG) is a 50 kDa glycoprotein expressed in the serum of various inflammatory diseases such as rheumatoid arthritis and inflammatory bowel disease. C-reactive protein (CRP) and serum amyloid A (SAA) are widely used as biomarkers to assess inflammation and primarily induced by IL-6, whereas LRG is induced not only by IL-6 but also by other inflammatory cytokines [10,11]. When patients are receiving IL-6 inhibitors such as tocilizumab, the inflammatory markers CRP and SAA do not increase even when inflammation is exacerbated [11]. Lipopolysaccharide (LPS) enhanced hepatic mRNA levels of LRG, SAA and serum amyloid P component, which is the mouse homolog of CRP [11]. LRG was highly expressed in the serum and GCF of patients with Stage III, Grade C periodontitis and decreased after completion of IPT [12]. Therefore, LRG could be useful as a clinical parameter for inflammatory conditions. The purpose of this study was to investigate the changes in clinical parameters of periodontitis and salivary LRG levels before and after IPT.

Materials and Methods

Study population

One hundred patients with chronic periodontitis who visited Igarashi Dental East Clinic from October 2021 to June 2022 were recruited to this study. They were divided into 50 Mild and 50 Severe groups based on the results of their periodontal examination, Mild group: Stage Ⅰ or Ⅱ, Grade A periodontitis and Severe group: Stage III, Grade B or C periodontitis. Seventeen of 50 patients in the Mild group, and 20 of 50 patients in the Severe group dropped out, leaving the Mild and Severe group with 33 and 30 patients, who were eventually followed up and analyzed (Fig. 1). All patients were confirmed to be systemically healthy and had no history of periodontal or antibiotic treatment in the 3 months prior to participation in this study. None of the patients were smokers. This study was conducted with the approval of Nihon University School of Dentistry at Matsudo Ethics Review Committee (EC 21-015A), and written informed consent was obtained from all patients. They received IPT, including oral hygiene instruction, scaling and root planing, and professional mechanical tooth cleaning. The average period of IPT was three months.

Fig. 1 Overview of the present study

(A) Flow diagram showing the progress of the study. Between October 2021 to June 2022, 100 chronic periodontitis patients were enrolled. They were divided into 50 Mild and 50 Severe groups based on the results of their periodontal examination, and subsequently received IPT, including oral hygiene instruction, scaling and root planing, and professional mechanical tooth cleaning. Seventeen of 50 patients in the Mild group, and 20 of 50 patients in the Severe group dropped out, leaving the Mild and Severe group with 33 and 30 patients, who were eventually followed up and analyzed. (B) Visit protocol. Clinical examinations were performed before and after IPT. Sampling of saliva was performed at the second visit and after IPT.

Sample size calculation

Sample size calculation of salivary LRG levels in this study was determined based on a study comparing LRG levels in GCF before and after treatment in 25 healthy patients and 25 patients with periodontitis [12]. The sample size was calculated using the statistical software G*Power 3.1.9.7 (Heinrich Heine University Düsseldorf, Düsseldorf, Germany) [13] and it was concluded that samples from 18 patients were required to reach a power of 80% at a significance level of 5% for both groups (Mild and Severe).

Clinical parameters

Periodontal tissue examinations were performed two times (before and after IPT) by one periodontal specialist (N.I.). PD and CAL measurements were performed using a periodontal probe (CP11 Color-Coded Probe, Hu-Friedy, Chicago, IL, USA) at a probing pressure of 20 to 25 g by 6-point method, and the presence or absence of bleeding after probing was recorded as BOP. BOP rate (%) was calculated by dividing the number of BOP sites by the number of measuring PD sites [14]. CAL values were the sum of the amount of gingival recession (distance from the cement-enamel junction to the marginal gingiva) and PD. PD and CAL were evaluated by summing the numerical values and dividing by the number of teeth to calculate the mean PD and CAL. In addition, O’Leary’s plaque control record (PCR) [15], PISA and periodontal epithelial surface area (PESA) were measured. PISA and PESA were calculated according to the method of Nesse et al. [7].

Collections of saliva samples

Saliva samples were collected twice (before and after IPT). All saliva collections were performed prior to oral hygiene. Saliva was collected by having patients chew paraffin wax (Oral Care, Tokyo, Japan) and the accumulated fluid was spat into a 50-mL centrifuge tube and saliva collection was performed until 5 mL with continuous spitting [16], and then stored at −80°C until the amount of LRG protein was measured.

Enzyme-linked immunosorbent assay (ELISA)

The amounts of LRG protein in saliva were measured by ELISA (Human LRG assay kit: Immuno-Biological Laboratories Co., Ltd, Fujioka, Japan) using anti-human LRG rabbit IgG [17]. Saliva samples were centrifuged (3,000 rpm, 15 min), and the supernatants were collected. Five µL of saliva was dissolved in 395 µL of sample dilution buffer in the assay kit. Diluted saliva samples (100 µL) were incubated in Human LRG antibody-precoated 96-well plates for 18 h, and after washing, HRP-conjugated secondary antibodies were added to the wells and incubated for 30 min at 37°C. After washing, 3, 3′, 5, 5′-tetramethylbenzidine dihydrochloride substrate solutions were added to the wells, protected from light, and incubated at room temperature for 30 min. Thereafter, color development was stopped with stop solution, and the absorbance of each well was measured at 450 nm using a microplate reader. All measurements were performed in duplicate, and the concentration of LRG was expressed in ng/mL.

Statistical analysis

Chi-square for independence test 2 × 2 contingency was used to determine whether there was a significant difference in the proportion of males and females in the Mild and Severe groups (Table 1). Differences in clinical parameters, age and number of teeth between the Mild and Severe groups were tested by Mann-Whitey U test and Wilcoxon signed-rank test and are presented as medians and interquartile ranges in Tables 1 and 2. Spearman's rank correlation was used for the correlation between salivary LRG levels and mean PD and CAL, BOP rate, PISA, PESA and PCR in Table 3. Significance differences in LRG expression levels between the Mild and Severe groups before and after IPT were determined by the Steel-Dwass test and presented as median and interquartile range, as this was a comparison of four groups (Fig. 2). The results were statistically significant when P < 0.05. Statcel: the useful addin forms on Excel, 4th ed. (OMS Ltd. Publisher, Tokyo, Japan) was used for statistical analyses.

Table 1 Patient characteristics

	Mild group (n = 33)	Severe group (n = 30)	P value
Gender (male/female)	13/20	13/17	0.757
Age	43.00 (27.00-55.00)	43.00 (27.00-55,00)	0.106
Number of teeth	28.00 (27.00-29.00)	28.00 (26.25-28.75)	0.454

Mild group: Stage Ⅰ or Ⅱ, Grade A periodontitis, Severe group: Stage III, Grade B or C periodontitis, Chi-square for independence test 2 × 2 contingency was used for gender, median and interquartile range were used for age and number of teeth. P < 0.05

 

Table 2 Changes in clinical parameters at Mild and Severe groups before and after IPT

		Mild group (n = 33)	Severe group (n = 30)	P value
Average PD (mm)	before IPT	2.50 (2.40-2.60)	3.10 (2.83-3.30)	<0.05
	after IPT	2.50 (2.40-2.60)	2.90 (2.70-3.00)	<0.05
	P value	0.426	<0.05
Average CAL (mm)	before IPT	2.96 (2.79-3.15)	4.09 (3.50-4.79)	<0.05
	after IPT	3.00 (2.82-3.27)	3.61 (3.33-3.92)	<0.05
	P value	0.461	<0.05
BOP rate (%)	before IPT	8.70 (3.8-16.8)	24.20 (14.2-41.5)	<0.05
	after IPT	5.00 (3.6-7.2)	10.80 (5.1-14.1)	<0.05
	P value	<0.05	<0.05
PISA (mm2)	before IPT	111.80 (53.90-230.60)	522.96 (259.00-809.90)	<0.05
	after IPT	81.00 (50.20-108.60)	177.85 (97.43-290.85)	<0.05
	P value	<0.05	<0.05
PESA (mm2)	before IPT	1,322.30 (1,259.80-1,386.50)	1,661.70 (1,526.88-1,800.38)	<0.05
	after IPT	1,309.60 (1,249.00-1,363.10)	1,543.00 (1,435.83-1,648.13)	<0.05
	P value	0.393	<0.05
PCR (%)	before	36.10 (17.0-47.3)	51.00 (35.7-67.8)	<0.05
	after	16.10 (5.4-27.7)	19.90 (11.2-34.7)	0.167
	P value	<0.05	<0.05

Mild group: Stage Ⅰ or Ⅱ, Grade A periodontitis, Severe group: Stage III, Grade B or C periodontitis, median and interquartile range (Mild group n = 33, Severe group n = 30), P < 0.05

 

Table 3 Correlation between salivary LRG and clinical parameters

	Average PD (mm)	Average CAL (mm)	BOP rate (%)	PISA (mm2)	PESA (mm2)	PCR (%)	Saliva LRG (ng/mL)
Saliva LRG (ng/mL)	0.37*	0.434*	0.326*	0.377*	0.405*	0.086	1

Statistically significant differences. (P < 0.05) by Spearman rank correlat

 

Fig. 2 Box plot showing changes of LRG in saliva at Mild and Severe groups before and after IPT (median and interquartile range; Mild group [n = 33], Severe group [n = 30]). P < 0.05

Results

Clinical parameters

Gender, age and number of teeth of the 63 patients are shown in Table 1. There were no significant differences in gender, age and number of teeth between the Mild and Severe group. Clinical parameters before and after IPT for the Mild and Severe group are shown in Table 2. Average PD, CAL, BOP rate, PISA, PESA and PCR of the Mild group at before IPT were 2.50 (2.40-2.60) mm, 2.96 (2.79-3.15) mm, 8.7 (3.8-16.8)%, 111.80 (53.90-230.60) mm², 1,322.30 (1,259.80-1,386.50) mm² and 36.1 (17.0-47.3)%, and 3.10 (2.83-3.30) mm, 4.09 (3.50-4.79) mm, 24.2 (14.2-41.5) %, 522.96 (259.00-809.90) mm², 1,661.70 (1,526.88-1,800.38) mm² and 51.0 (35.7-67.8)% in the Severe group. All clinical parameters at before IPT showed significantly higher values in the Severe group compared to the Mild group. After IPT, BOP rate, PISA and PCR of the Mild group were significantly reduced to 5.00 (3.6-7.2) %, 81.00 (50.20-108.60) mm² and 16.1 (5.4-27.7) %. After IPT in the Severe group, mean PD, CAL, BOP rate, PISA, PESA and PCR were significantly reduced to 2.90 (2.70-3.00) mm, 3.61 (3.33-3.92) mm, 10.8 (5.1-14.1) %, 177.85 (97.43-290.85) mm², 1,543.00 (1,435.83-1,648.13) mm² and 19.9 (11.2-34.7) %.

Expression levels of LRG protein

The amount of LRG in saliva collected from the Mild and Severe groups before and after IPT is shown in Fig. 2. The median salivary LRG content before IPT was higher in the Severe group than in the Mild group, with a significant difference between the two groups. In addition, the amount of LRG decreased significantly in the Severe group after the IPT. In the Mild group, LRG protein levels did not change significantly after the IPT.

Correlation between salivary LRG and clinical parameters

Positive correlations were found between salivary LRG protein levels and mean PD, mean CAL, BOP rate, PISA and PESA. The strongest correlation between salivary LRG protein levels and mean CAL was observed (Table 3).

Discussion

To develop inflammatory biomarkers, proteomic analysis was utilized, and LRG was identified as a marker for evaluating disease activity in autoimmune diseases [18]. Like other acute-phase proteins, LRG is synthesized in hepatocytes and its production is upregulated in response to systemic inflammation [11]. However, LRG production is detected in hepatocytes and other cells such as neutrophils, macrophages and epithelial cells of inflamed tissues [19]. Furthermore, LRG induction is dependent not only on IL-6 but also on other inflammatory cytokines such as IL-1β, IL-22 and TNF-α [10]. These characteristics suggest that LRG more sensitively reflect inflammation than CRP, as CRP is primarily produced in the liver in the presence of IL-6. Therefore, LRG could be superior to CRP in evaluating mucosal healing in ulcerative colitis patients [20] and biomarker for disease activity during IL-6 blockade therapy of rheumatoid arthritis [21]. High-sensitivity CRP (hsCRP) is listed as a risk factor for systemic effects of periodontitis in the new grading classification of periodontitis formulated at the 2017 World Workshop on the Classification of Periodontal and Peri-implant Diseases and Conditions [22]. However, since blood sampling is required to measure hsCR, it is difficult to actively utilize it in daily dental practice. Therefore, as a simpler method, measuring inflammatory factors in saliva might be an alternative method for evaluating periodontitis. In the new stage and grade classification of periodontitis, the stage represents the destruction of periodontium and the grade represents the rate of progression, so it is difficult to analyze the results of this study using the grade classification. A study that evaluated the relationship between CRP in saliva and blood showed that CRP in saliva did not correlate with serum concentrations of CRP [23]. Studies on salivary LRG have shown that significantly higher mean levels of salivary LRG in the pediatric acute appendicitis group than in the control group [24]. Salivary levels of LRG were significantly higher in Stage Ⅲ periodontitis group [316.57 (61.34-1,097.64) ng/mL] than in periodontally healthy group [33.22 (3.1-209.26) ng/mL] [25]. These results demonstrate that salivary LRG is a useful inflammatory biomarker that reflects systemic effects. In this study, stimulated saliva was collected by chewing paraffin wax, but did not measure the salivary flow rate [16]. The effect of salivary flow rate on the saliva LRG levels remains a topic for future research. It has been reported that IL-1β and heat shock protein 70 levels in GCF are high in patients with periodontitis and decrease after IPT [9,26]. Therefore, there are other candidates that could be used as biomarkers for periodontitis besides salivary LRG. Therefore, it may be possible to predict the prognosis of periodontitis with even greater accuracy by combining salivary LRG with other biomarkers. Plaque index and PD are statistically associated with high body mass index (BMI) [27]. The limitation of this study is that it does not include information on the relationship between BMI and saliva LRG.

In this study, salivary LRG levels in the Severe group before IPT were significantly higher values than in the Mild group, and the salivary LRG levels in the Severe group significantly decreased after IPT (Fig. 2). Furthermore, among clinical parameters, there was the highest correlation between salivary LRG protein levels and mean CAL, suggesting a close relationship with periodontal tissue destruction. In addition, after IPT, BOP rate in the Severe group decreased to almost the same level as that in the Mild group before IPT, and there was no significant difference in the salivary LRG protein levels between the Mild and Severe groups, suggesting that the decrease in LRG levels is associated with improvement in gingival inflammation. These results suggest that the amount of salivary LRG might be a crucial biomarker for predicting the prognosis of periodontal therapy.

Abbreviations

BOP: bleeding on probing; CAL: clinical attachment level; CRP: C-reactive protein; ELISA: enzyme-linked immunosorbent assay; GCF: gingival crevicular fluid; GI: gingival index; hsCRP: high-sensitivity CRP; IL-6: interleukin-6; IPT: initial periodontal therapy; LRG: Leucine-rich α2 glycoprotein; PCR: O’Leary’s plaque control record; PD: probing depth; PESA: periodontal epithelial surface area; PISA: periodontal inflamed surface area; qPCR: quantitative polymerase chain reaction; SAA: serum amyloid A

Ethical Statements

This study was conducted with the approval of Nihon University Matsudo School of Dentistry Ethics Review Committee (EC 21-015A).

Conflicts of Interest

The authors have no potential conflicts of interest to report.

Funding

This study was supported in part by the Japan Society for the Promotion of Science KAKENHI Grants and Scientific Research (C); No. 18K09583 and 21K09922 to HT, 17K11994, 20K09945 and 23K09173 to YO, and a Nihon University Multidisciplinary Research Grant for 2021.

Author Contributions

NI: investigation; HT: conceptualization, formal analysis, methodology, investigation original draft writing; YO: conceptualization, resources, formal analysis, review, editing, supervision.

ORCID iD

¹⁾NI: [e-mail], https://orcid.org/0009-0006-8563-8223

^1,2)HT: [e-mail], https://orcid.org/0000-0002-2609-3052

^1,2)YO*: [e-mail], https://orcid.org/0000-0002-2767-844X

Data Availability Statements

All authors gave approval for the data in this study to be available.

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