J Periodontol • December 2014
Glycated Albumin and Calprotectin Levels in Gingival Crevicular Fluid From Patients With Periodontitis and Type 2 Diabetes Yukari Kajiura,* Mika Bando,* Yuji Inagaki,* Toshihiko Nagata,* and Jun-ichi Kido*
Background: Patients with diabetes mellitus (DM) have a high prevalence of periodontitis. Periodontitis in these patients is characterized by severe inflammation and tissue breakdown, and its diagnosis is important for cures of periodontitis and DM. The purpose of this study is to investigate the levels of glycated albumin (GA), a DM marker, and calprotectin, an inflammatory marker, in gingival crevicular fluid (GCF) from patients with periodontitis and DM (DM-P). Methods: The 78 participants in this study were patients with DM, chronic periodontitis (CP), DM-P, and healthy individuals (H). GCF and blood were collected, and GA and calprotectin in GCF were analyzed using Western blotting and enzyme-linked immunosorbent assay. Levels were compared among H, DM, CP, and DM-P groups. Blood GA and glycated hemoglobin (HbA1c) were measured, and the correlation among GCF GA and blood HbA1c or GA levels was investigated. Receiver operating characteristic (ROC) analysis for GCF GA to predict DM was performed. Results: GA was identified in GCF, and its amount/ concentration in GCF samples from DM and DM-P were significantly higher than those of non-DM groups (H and CP). Calprotectin amounts in GCF from CP and DM-P were significantly higher than in H and DM groups. GCF GA level was positively correlated with blood HbA1c and GA level. ROC analysis of GCF GA showed an optimal cutoff value to predict DM. Conclusions: GA showed a high level in GCF from patients with DM. Examination of GA and calprotectin in GCF may be useful for predicting DM-P. J Periodontol 2014;85: 1667-1675. KEY WORDS Calprotectin; diabetes mellitus; diagnosis; gingival crevicular fluid; glycated albumin; periodontics. * Department of Periodontology and Endodontology, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima, Japan.
P
eriodontitis is an oral inflammatory disease with a high incidence in middle-aged and elderly people.1,2 Diabetes mellitus (DM) is a major metabolic disorder with abnormal glucose metabolism; its prevalence is increasing in many countries.2,3 DM causes inflammatory complications, including diabetic nephropathy, neuropathy, and retinopathy, and is associated with cardiovascular diseases, vascular abnormalities in the brain, and atherosclerosis.4-7 Periodontitis is a common complication of DM, and its incidence in patients with DM is higher than in the general population.8-10 Periodontitis in patients with DM often shows edematous gingival swelling with severe inflammation and breakdown of periodontal tissues.11,12 The hyperglycemia in DM induces the production of glycated proteins known as advanced glycation end products (AGEs) and results in diabetic complications. AGEs cause vascular abnormalities, altered collagen metabolism, and dysfunction of immune cells and regulate the expression of inflammatory cytokines and chemokines in periodontal tissues.13-16 Excess AGEs accumulate in the periodontal tissues of patients with DM and aggravate periodontal diseases.17,18 On the other hand, periodontitis also appears to affect DM,3 and treatments of periodontitis such as scaling and root planing and administration of antibiotics modestly improved DM doi: 10.1902/jop.2014.140241
1667
Glycated Albumin in Gingival Crevicular Fluid from Patients With Diabetes
condition 3 to 4 months after periodontal therapies.19 The diagnosis of periodontitis in patients with DM may contribute to the prediction of aggravation of periodontitis and screening of undiagnosed DM at a dental office. DM is medically diagnosed by determining glycated hemoglobin (HbA1c), glycated albumin (GA), and blood glucose levels. HbA1c and GA are intermediate products called Amadori compounds, involved in the non-enzymatic glycation reactions of blood proteins, which finally change to AGEs.20,21 HbA1c and GA levels reflect the status of glycemic control, and these variables were shown to be positively correlated; the level of GA or HbA1c was also significantly associated with plasma glucose levels in individuals with and without DM in epidemiologic research.22,23 The HbA1c level represents the mean glycemic status over the previous 2 to 3 months but does not reflect glycemic condition in the short term or provide an accurate measure in patients with anemia and hemolysis.24,25 In contrast, GA more exactly shows the glycemic condition in the short term (2 to 3 weeks) because the half-life of albumin is approximately 17 days.22,23,26,27 GA has been used to evaluate postprandial glycemic status and glycemic control after medication for DM treatment and accurately reflects the status of glycemic control in patients with DM with certain diseases, including anemia, hemoglobin disorder, and renal failure, as well as in pregnancy,26,27 suggesting that GA is a reliable marker to evaluate glycemic status in patients with DM. Calprotectin is a major cytosolic protein in leukocytes and is also identified in monocytes/macrophages and epithelial cells.28 Calprotectin levels in plasma, synovial fluid, or feces from patients with inflammatory diseases, including cystic fibrosis, rheumatoid arthritis, and ulcerative colitis, are increased in comparison with those in healthy patients.29-31 Calprotectin was also detected in gingival crevicular fluid (GCF), and its level in GCF samples from patients with periodontitis was significantly higher than in those without periodontitis.32-35 Calprotectin levels in GCF from patients with periodontitis were also decreased by periodontal treatments and predicted periodontal disease activity in patients with periodontitis,36,37 suggesting that calprotectin in GCF is a useful marker for periodontal diseases. The authors of the present study recently identified GA in GCF samples from patients with DM and periodontitis (DM-P). The purpose of this study is to compare the levels of GA and calprotectin in GCF samples from individuals with or without periodontitis and DM, and to investigate the possibility that GA and calprotectin in GCF are markers that can predict periodontitis in patients with DM. 1668
Volume 85 • Number 12
MATERIALS AND METHODS Participants and Clinical Examinations This clinical study was performed from March 2012 to February 2014 with the approval of the Ethics Committee of Tokushima University Hospital, Tokushima, Japan) in accordance with the Helsinki Declaration of 1975, as revised in 2000. Seventyeight individuals (33 males and 45 females, aged 31 to 84 years; mean age: 65.8 – 8.7 years) gave written informed consent after hearing an explanation of the present study. Although all participants gave their consent for collection of GCF and medical information, only 50 of them agreed to blood collection. Patients with periodontitis and type 2 DM as well as healthy volunteers without these two diseases were recruited from Tokushima University Hospital and the University of Tokushima Graduate School. All participants were non-smokers, and none of them had used antibiotic therapies within 3 months. The participants were classified into four groups: DM without periodontitis (DM), chronic periodontitis without DM (CP), periodontitis with DM (DM-P), and no DM and no periodontitis (healthy [H]); their characteristics are shown in Table 1. Probing depth (PD), clinical attachment level (CAL), and gingival index (GI) were examined as the clinical periodontal indicators. Periodontitis was evaluated at 219 sites by two clinical indicators: PD >4 mm and GI score >1. GI score was examined according to the ¨ e and Silness.38 DM was defined as standard of Lo HbA1c >6.5% (National Glycohemoglobin Standardization Program [NGSP]).24 Blood samples were collected from 50 individuals with or without DM using sterilized disposable needles and syringes, and HbA1c and GA percentages in blood samples were determined by a company specializing in clinical examinations.† GCF Sampling GCF was collected using paper strips‡ according to previously described methods.32-34 Briefly, oral sites for GCF sampling were isolated with cotton rolls and gently air-dried after the supragingival plaque had been removed. A paper strip was inserted into a gingival crevice without periodontitis or a periodontal pocket with periodontitis and held there for 10 seconds. GCF collection was sequentially repeated three times using three strips. The GCF volume in strip paper was determined using a calibrated unit,§ and the total volume from one site was calculated. GCF was extracted from a phosphate buffer solution (pH = 7.4) with 0.2 mM phenylmethylsulfonyl fluoride by centrifugation according to a modified † Bio Medical Laboratories, Tokyo, Japan. ‡ PerioPaper, Oraflow, Smithtown, NY. § Periotron 8000, Harco Electronics, Winnipeg, MB, Canada.
Kajiura, Bando, Inagaki, Nagata, Kido
J Periodontol • December 2014
Table 1.
Characteristics of Participants and GCF Samples Characteristic
H
DM
CP
DM-P
n
25
10
23
20
8:17
5:5
11:12
9:11
Sex (n, males:females) Age (years)
66.7 – 10.9
65.5 – 6.1
65.2 – 9.1
65.9 – 8.8
GCF sampling n Volume (mL)
60 0.88 – 0.64
32 1.04 – 0.75
61 2.56 – 1.00*†
66 5.32 – 1.73*†
PD (mm)
2.02 – 0.68
2.19 – 0.59
5.44 – 1.73*†
5.32 – 1.73*†
CAL (mm)
2.45 – 0.93
3.25 – 2.00*
6.14 – 1.99*†
6.22 – 2.03*†
0
0
1.69 – 0.56*†
1.61 – 0.60*†
GI
Data are mean – SD unless otherwise noted. * Statistically significant difference from H (P
Glycated Albumin and Calprotectin Levels in Gingival Crevicular Fluid From Patients With Periodontitis and Type 2 Diabetes Yukari Kajiura,* Mika Bando,* Yuji Inagaki,* Toshihiko Nagata,* and Jun-ichi Kido*
Background: Patients with diabetes mellitus (DM) have a high prevalence of periodontitis. Periodontitis in these patients is characterized by severe inflammation and tissue breakdown, and its diagnosis is important for cures of periodontitis and DM. The purpose of this study is to investigate the levels of glycated albumin (GA), a DM marker, and calprotectin, an inflammatory marker, in gingival crevicular fluid (GCF) from patients with periodontitis and DM (DM-P). Methods: The 78 participants in this study were patients with DM, chronic periodontitis (CP), DM-P, and healthy individuals (H). GCF and blood were collected, and GA and calprotectin in GCF were analyzed using Western blotting and enzyme-linked immunosorbent assay. Levels were compared among H, DM, CP, and DM-P groups. Blood GA and glycated hemoglobin (HbA1c) were measured, and the correlation among GCF GA and blood HbA1c or GA levels was investigated. Receiver operating characteristic (ROC) analysis for GCF GA to predict DM was performed. Results: GA was identified in GCF, and its amount/ concentration in GCF samples from DM and DM-P were significantly higher than those of non-DM groups (H and CP). Calprotectin amounts in GCF from CP and DM-P were significantly higher than in H and DM groups. GCF GA level was positively correlated with blood HbA1c and GA level. ROC analysis of GCF GA showed an optimal cutoff value to predict DM. Conclusions: GA showed a high level in GCF from patients with DM. Examination of GA and calprotectin in GCF may be useful for predicting DM-P. J Periodontol 2014;85: 1667-1675. KEY WORDS Calprotectin; diabetes mellitus; diagnosis; gingival crevicular fluid; glycated albumin; periodontics. * Department of Periodontology and Endodontology, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima, Japan.
P
eriodontitis is an oral inflammatory disease with a high incidence in middle-aged and elderly people.1,2 Diabetes mellitus (DM) is a major metabolic disorder with abnormal glucose metabolism; its prevalence is increasing in many countries.2,3 DM causes inflammatory complications, including diabetic nephropathy, neuropathy, and retinopathy, and is associated with cardiovascular diseases, vascular abnormalities in the brain, and atherosclerosis.4-7 Periodontitis is a common complication of DM, and its incidence in patients with DM is higher than in the general population.8-10 Periodontitis in patients with DM often shows edematous gingival swelling with severe inflammation and breakdown of periodontal tissues.11,12 The hyperglycemia in DM induces the production of glycated proteins known as advanced glycation end products (AGEs) and results in diabetic complications. AGEs cause vascular abnormalities, altered collagen metabolism, and dysfunction of immune cells and regulate the expression of inflammatory cytokines and chemokines in periodontal tissues.13-16 Excess AGEs accumulate in the periodontal tissues of patients with DM and aggravate periodontal diseases.17,18 On the other hand, periodontitis also appears to affect DM,3 and treatments of periodontitis such as scaling and root planing and administration of antibiotics modestly improved DM doi: 10.1902/jop.2014.140241
1667
Glycated Albumin in Gingival Crevicular Fluid from Patients With Diabetes
condition 3 to 4 months after periodontal therapies.19 The diagnosis of periodontitis in patients with DM may contribute to the prediction of aggravation of periodontitis and screening of undiagnosed DM at a dental office. DM is medically diagnosed by determining glycated hemoglobin (HbA1c), glycated albumin (GA), and blood glucose levels. HbA1c and GA are intermediate products called Amadori compounds, involved in the non-enzymatic glycation reactions of blood proteins, which finally change to AGEs.20,21 HbA1c and GA levels reflect the status of glycemic control, and these variables were shown to be positively correlated; the level of GA or HbA1c was also significantly associated with plasma glucose levels in individuals with and without DM in epidemiologic research.22,23 The HbA1c level represents the mean glycemic status over the previous 2 to 3 months but does not reflect glycemic condition in the short term or provide an accurate measure in patients with anemia and hemolysis.24,25 In contrast, GA more exactly shows the glycemic condition in the short term (2 to 3 weeks) because the half-life of albumin is approximately 17 days.22,23,26,27 GA has been used to evaluate postprandial glycemic status and glycemic control after medication for DM treatment and accurately reflects the status of glycemic control in patients with DM with certain diseases, including anemia, hemoglobin disorder, and renal failure, as well as in pregnancy,26,27 suggesting that GA is a reliable marker to evaluate glycemic status in patients with DM. Calprotectin is a major cytosolic protein in leukocytes and is also identified in monocytes/macrophages and epithelial cells.28 Calprotectin levels in plasma, synovial fluid, or feces from patients with inflammatory diseases, including cystic fibrosis, rheumatoid arthritis, and ulcerative colitis, are increased in comparison with those in healthy patients.29-31 Calprotectin was also detected in gingival crevicular fluid (GCF), and its level in GCF samples from patients with periodontitis was significantly higher than in those without periodontitis.32-35 Calprotectin levels in GCF from patients with periodontitis were also decreased by periodontal treatments and predicted periodontal disease activity in patients with periodontitis,36,37 suggesting that calprotectin in GCF is a useful marker for periodontal diseases. The authors of the present study recently identified GA in GCF samples from patients with DM and periodontitis (DM-P). The purpose of this study is to compare the levels of GA and calprotectin in GCF samples from individuals with or without periodontitis and DM, and to investigate the possibility that GA and calprotectin in GCF are markers that can predict periodontitis in patients with DM. 1668
Volume 85 • Number 12
MATERIALS AND METHODS Participants and Clinical Examinations This clinical study was performed from March 2012 to February 2014 with the approval of the Ethics Committee of Tokushima University Hospital, Tokushima, Japan) in accordance with the Helsinki Declaration of 1975, as revised in 2000. Seventyeight individuals (33 males and 45 females, aged 31 to 84 years; mean age: 65.8 – 8.7 years) gave written informed consent after hearing an explanation of the present study. Although all participants gave their consent for collection of GCF and medical information, only 50 of them agreed to blood collection. Patients with periodontitis and type 2 DM as well as healthy volunteers without these two diseases were recruited from Tokushima University Hospital and the University of Tokushima Graduate School. All participants were non-smokers, and none of them had used antibiotic therapies within 3 months. The participants were classified into four groups: DM without periodontitis (DM), chronic periodontitis without DM (CP), periodontitis with DM (DM-P), and no DM and no periodontitis (healthy [H]); their characteristics are shown in Table 1. Probing depth (PD), clinical attachment level (CAL), and gingival index (GI) were examined as the clinical periodontal indicators. Periodontitis was evaluated at 219 sites by two clinical indicators: PD >4 mm and GI score >1. GI score was examined according to the ¨ e and Silness.38 DM was defined as standard of Lo HbA1c >6.5% (National Glycohemoglobin Standardization Program [NGSP]).24 Blood samples were collected from 50 individuals with or without DM using sterilized disposable needles and syringes, and HbA1c and GA percentages in blood samples were determined by a company specializing in clinical examinations.† GCF Sampling GCF was collected using paper strips‡ according to previously described methods.32-34 Briefly, oral sites for GCF sampling were isolated with cotton rolls and gently air-dried after the supragingival plaque had been removed. A paper strip was inserted into a gingival crevice without periodontitis or a periodontal pocket with periodontitis and held there for 10 seconds. GCF collection was sequentially repeated three times using three strips. The GCF volume in strip paper was determined using a calibrated unit,§ and the total volume from one site was calculated. GCF was extracted from a phosphate buffer solution (pH = 7.4) with 0.2 mM phenylmethylsulfonyl fluoride by centrifugation according to a modified † Bio Medical Laboratories, Tokyo, Japan. ‡ PerioPaper, Oraflow, Smithtown, NY. § Periotron 8000, Harco Electronics, Winnipeg, MB, Canada.
Kajiura, Bando, Inagaki, Nagata, Kido
J Periodontol • December 2014
Table 1.
Characteristics of Participants and GCF Samples Characteristic
H
DM
CP
DM-P
n
25
10
23
20
8:17
5:5
11:12
9:11
Sex (n, males:females) Age (years)
66.7 – 10.9
65.5 – 6.1
65.2 – 9.1
65.9 – 8.8
GCF sampling n Volume (mL)
60 0.88 – 0.64
32 1.04 – 0.75
61 2.56 – 1.00*†
66 5.32 – 1.73*†
PD (mm)
2.02 – 0.68
2.19 – 0.59
5.44 – 1.73*†
5.32 – 1.73*†
CAL (mm)
2.45 – 0.93
3.25 – 2.00*
6.14 – 1.99*†
6.22 – 2.03*†
0
0
1.69 – 0.56*†
1.61 – 0.60*†
GI
Data are mean – SD unless otherwise noted. * Statistically significant difference from H (P
