Correlation Between Arteriosclerosis and Periodontal Condition Assessed by Lactoferrin and α1-Antitrypsin Levels in Gingival Crevicular Fluid Shuji Hayashi,1 MD, Hirotsugu Yamada,2 MD, Makoto Fukui,3 DDS, Hiro-o Ito,3 DDS, and Masataka Sata,2 MD Summary Patients with periodontal disease exhibit exacerbated atherosclerosis, aortic stiffness, or vascular endothelial dysfunction. However, in a recent scientific statement, the American Heart Association noted that neither has periodontal disease been proven to cause atherosclerotic vascular disease nor has the treatment of periodontal disease been proven to prevent atherosclerotic vascular disease. Therefore, the aim of the present study was to examine the correlation between periodontal condition and arteriosclerosis in patients with coronary artery disease (CAD), which is usually accompanied by systemic arteriosclerosis. We measured levels of gingival crevicular fluid lactoferrin (GCF-Lf) and α1-antitrypsin (GCF-AT) in 72 patients (67 ± 8 years, 56 men) with CAD. Furthermore, we evaluated the maximum intima–media thickness (max IMT) and plaque score of the carotid arteries as well as brachial–ankle pulse wave velocity (baPWV) and flow-mediated dilation (FMD) of the brachial artery, each of which is a parameter for determining arteriosclerosis status. The average level of GCF-Lf was 0.29 ± 0.36 µg/mL and that of GCF-AT was 0.31 ± 0.66 µg/mL, with significant correlation between the two (r = 0.701, P < 0.001). No significant difference in GCF-Lf and GCF-AT levels was observed between patients with single-, double-, and triple-vessel CAD. There were no significant correlations between the arteriosclerosis parameters (ie, max IMT, plaque score, baPWV, and FMD) and GCF-Lf or GCF-AT. No correlation between the GCF biomarkers and the severity of arteriosclerosis was detected. This result may suggest that worsening of the periodontal condition assessed by GCF biomarkers is not a major potential risk factor for arteriosclerosis. (Int Heart J 2015; 56: 000-000) Key words: Periodontal disease, Coronary artery disease, Atherosclerosis
A
correlation has been reported between periodontal disease and atherosclerotic vascular disease.1,2) Some investigators have reported that patients with periodontal disease exhibit exacerbated atherosclerosis, aortic stiffness, or vascular endothelial dysfunction.3-5) However, in a recent scientific statement, the American Heart Association noted that neither has periodontal disease been proven to cause atherosclerotic vascular disease nor has the treatment of periodontal disease been proven to prevent atherosclerotic vascular disease.1) Therefore, the aim of the present study was to examine the correlation between periodontal condition and arteriosclerosis in patients with coronary artery disease (CAD), which is usually accompanied by systemic arteriosclerosis. There are several methods to evaluate periodontal condition in clinical settings. In this study, we measured inflammatory biomarker levels of gingival crevicular fluid (GCF), a tissue fluid extravasated from the crevicular epithelium. Because periodontal inflammatory conditions cause alterations in GCF inflammation biomarkers, these conditions can be assessed by
investigating the biomarkers.6-8) This method has the advantage that the periodontal condition can be quantified by internists without expertise in periodontal disease. We investigated the correlation between the GCF biomarker levels and non-invasive parameters of arteriosclerosis status such as carotid artery intima–media thickness, brachial–ankle pulse wave velocity (baPWV), and brachial artery flow-mediated dilation (FMD)9,10) in patients with CAD.
Methods Subjects: Among patients hospitalized between April 2011
and July 2012 to undergo coronary angiography or percutaneous coronary intervention for known CAD at the Department of Cardiovascular Medicine, Tokushima University Hospital, 80 patients aged between 20 and 75 years with at least 10 teeth remaining were randomly selected and enrolled in this study. Patients with CAD were defined as those with a history of cor-
From the 1 Ultrasound Examination Center, 2 Department of Cardiovascular Medicine, Tokushima University Hospital, and 3 Department of Preventive Dentistry, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima, Japan. Address for correspondence: Hirotsugu Yamada, MD, Department of Cardiovascular Medicine, Tokushima University Hospital, 2-50-1 Kuramoto, Tokushima 7708504, Japan. E-mail:
[email protected] Received for publication June 6, 2015. Revised and accepted June 19, 2015. Released in advance online on J-STAGE November 9, 2015. (This is “Advance Publication”.) All rights reserved by the International Heart Journal Association. 1
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onary revascularization or scheduled for coronary revascularization according to standard protocol.11,12) Eight patients were excluded because of malignant tumor comorbidity, consumption of corticosteroids or immunosuppressants, immunodeficiency diseases, undergoing dialysis, or liver cirrhosis. Hypertension was defined as a systolic blood pressure of ≥ 140 mmHg, a diastolic blood pressure of ≥ 90 mmHg, or the use of antihypertensive drug treatment. Dyslipidemia was defined as a serum concentration level of low-density lipoprotein cholesterol (LDL-C) ≥ 140 mg/dL, that of triglycerides ≥ 150 mg/dL, a serum concentration of high-density lipoprotein cholesterol (HDL-C) < 40 mg/dL, or antihyperlipidemic drug treatment. Diabetes mellitus was defined as a fasting plasma glucose level of ≥ 126 mg/dL, a 2-hour plasma glucose level after a 75-g oral glucose load of ≥ 200 mg/dL, hemoglobin A1c level of ≥ 6.5%, or the use of antidiabetic medication. Chronic renal failure was defined as an estimated glomerular filtration rate of < 60 mL/ minute/1.73 m2. The Brinkman index was defined as the number of cigarettes smoked per day multiplied by the number of years of smoking. GCF biomarker level examination: On day 1 of hospitalization, 72 patients eligible for analysis underwent GCF biomarker level testing. The test involved collecting GCF with a brush (Perio-catcher, Ikagaku Company, Limited, Kyoto, Japan) and measuring GCF lactoferrin (GCF-Lf) and α1-antitrypsin (GCF-AT) levels. Because lactoferrin is an iron-binding protein that exhibits bactericidal action, GCF-Lf increases with increasing periodontal inflammation.8) Similarly, GCF-AT increases when periodontal inflammation is increased because α1-antitrypsin is a major inhibitor of serine proteases.7) The subjects were instructed to refrain from eating, smoking, brushing their teeth, and gargling for at least 30 minutes before the sample collection. GCF samples were collected from the gingival crevices of 10 teeth between the left and right maxillary second premolars, taking advantage of the capillary phenomenon using a small brush of 2-mm diameter and 6-mm length. The brush was lightly and smoothly traced back and forth across the border between the teeth and gums for 30 seconds. In the case of subjects who had missing teeth among the 10 target teeth, other teeth were chosen to obtain 10 teeth in total. This test has been offered as a diagnostic kit for periodontal disease by Ikagaku. The results for both GCF-Lf and GCF-AT levels were reported as negative (< 0.40 µg/mL), weak positive (0.40–0.79 µg/mL), or strong positive (≥ 0.8 µg/mL), and in the analysis, we used this criterion as the severity of periodontal condition. Blood and arteriosclerosis examination: On day 1 of hospitalization, subjects underwent blood testing, carotid ultrasonography, and measurement of baPWV and FMD. These tests were performed according to standard protocols after ensuring that patients were in a restful state. Blood tests were performed before breakfast. Serum LDL-C, HDL-C, triglycerides, malondialdehyde-modified low-density lipoprotein (MD-LDL), creatinine, and plasma brain natriuretic peptide levels were measured. Maximum intima–media thickness (max IMT) and plaque score for the carotid arteries were assessed by high-end ultrasound systems (LOGIQ 7, GE Healthcare, Milwaukee, Wisconsin, USA; ProSound α-10, Hitachi-Aloka Medical, Tokyo) in accordance with the standard protocol.13) Max IMT was obtained for the posterior wall of the internal carotid artery, bulbus, and common carotid artery on both the right and
left sides. Plaque score was defined as the sum of all plaque thicknesses for the internal carotid artery, bulbus, and common carotid artery on both the right and left sides. We measured baPWV by BP-203 RPE III (OMRON COLIN Company, Limited, Tokyo); the mean value of the baPWV for the right and left sides was used in the statistical analysis. FMD was measured using UNEX-EF18G (UNEX Corporation, Aichi, Japan) in accordance with the standard protocol.14) On the day of the FMD examination, the patients were requested to fast and abstain from smoking and consuming caffeine-containing beverages. Only drinking water was given to the subjects, and all medications were withheld. The FMD measurement was performed between 8:30 and 9:30. The protocol was approved by the Ethics Committee of Tokushima University Hospital and informed consent was obtained from all subjects. Statistical analysis: Data analysis was performed using SPSS software, version 19.0 (IBM Corporation, Armonk, NY, USA). Data are presented as the mean ± standard deviation (SD) for continuous variables and percentages for categorical variables. Comparisons among 3 groups were analyzed by the Tukey– Kramer test or the Kruskal–Wallis test. Correlations between GCF-Lf, GCF-AT, and other indices were analyzed using the Pearson method. Statistical significance was set at P < 0.05.
Results The patient characteristics are presented in Table I. None of the patients were current smokers. LDL-C level was maintained at good levels (90 ± 25 mg/dL), which may be attributed in part to the use of statins by 68 (94.4%) patients. However, Table I. Patient Characteristics Variable Clinical characteristic Age (years) Male, n (%) Brinkman index Hypertension, n (%) Dyslipidemia, n (%) Diabetes mellitus, n (%) Chronic renal failure, n (%) Blood examination LDL-C (mg/dL) Triglycerides (mg/dL) HDL-C (mg/dL) MDA-LDL (U/L) Hemoglobin A1c (%) Brain natriuretic peptide (pg/mL) GCF biomarker level examination Lactoferrin (µg/mL) α1-Antitrypsin (µg/mL) Arteriosclerosis examination baPWV (cm/s) max IMT (mm) Plaque score FMD (%)
Value 67 ± 8 56 (77.8) 705 ± 674 61 (84.7) 69 (95.8) 39 (54.2) 37 (51.4) 90 ± 25 111 ± 56 54 ± 17 106 ± 35 6.2 ± 1.0 84.2 ± 85.9 0.29 ± 0.36 0.31 ± 0.66 1635 ± 299 2.5 ± 0.9 11.7 ± 7.7 4.3 ± 2.1
Values are the mean ± standard deviation or number of subjects (%). LDLC indicates low-density lipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol; MDA-LDL, malondialdehyde-modified low-density lipoprotein; GCF, gingival crevicular fluid; max IMT, maximum intimamedia thickness; baPWV, brachial-ankle pulse wave velocity; and FMD, flow mediated dilation.
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PERIODONTAL CONDITION AND ARTERIOSCLEROSIS Table II. Comparison of GCF-Lf and GCF-AT Levels Based on the Number of Coronary Lesions
GCF-Lf (µg/mL) GCF-AT (µg/mL)
1-VD (n = 28)
2-VD (n = 28)
3-VD (n = 16)
1-VD versus 2-VD P
1-VD versus 3-VD P
2-VD versus 3-VD P
0.35 ± 0.38 0.42 ± 0.90
0.26 ± 0.37 0.19 ± 0.20
0.25 ± 0.28 0.35 ± 0.72
0.570 0.386
0.653 0.934
1.000 0.717
Data are expressed as the mean ± standard deviation. GCF indicates gingival crevicular fluid; Lf, lactoferrin; AT, α1-antitrypsin; and VD, vessel disease.
Table III. Correlations Between Parameters Related to Arteriosclerosis and GCF-Lf or GCF-AT Variable GCF-Lf LDL-C (mg/dL) Triglycerides (mg/dL) HDL-C (mg/dL) MDA-LDL (U/L) Creatinine (mg/dL) Brain natriuretic peptide (pg/mL) Number of coronary lesions max IMT (mm) Plaque score baPWV (cm/s) FMD (%) GCF-AT LDL (mg/dL) Triglycerides (mg/dL) HDL (mg/dL) MDA-LDL (U/L) Creatinine (mg/dL) Brain natriuretic peptide (pg/mL) Number of coronary lesions max IMT (mm) Plaque score baPWV (cm/s) FMD (%)
Correlation coefficient
P
–0.142 –0.051 0.034 –0.035 –0.173 –0.163 –0.118 0.006 –0.009 –0.004 –0.116
0.234 0.672 0.776 0.773 0.146 0.171 0.322 0.963 0.942 0.972 0.466
–0.108 0.019 –0.038 –0.003 –0.134 –0.078 –0.068 0.024 0.096 0.023 –0.083
0.365 0.873 0.754 0.979 0.262 0.515 0.571 0.849 0.449 0.854 0.601
GCF indicates gingival crevicular fluid; Lf, lactoferrin; AT, α1-antitrypsin; LDL-C, low-density lipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol; MDA-LDL, malondialdehyde-modified low-density lipoprotein; max IMT, maximum intima-media thickness; baPWV, brachial-ankle pulse wave velocity; and FMD, flow-mediated dilation.
max IMT (2.5 ± 0.9 mm) and plaque scores (11.7 ± 7.7) were increased, which indicated that atherosclerosis was progressing in our subjects. A baPWV of 1635 ± 299 cm/s also indicated increased aortic stiffness. Impairment of FMD (4.3 ± 2.1%) showed that the subjects had vascular endothelial dysfunction. GCF-Lf was 0.29 ± 0.36 µg/mL and GCF-AT was 0.31 ± 0.66 µg/mL, with a significant correlation between the two (r = 0.701, P < 0.001). Table II shows a comparison of GCF-Lf and -AT levels based on the number of coronary lesions. No significant difference was observed between the 3 groups regarding GCF-Lf and GCF-AT levels. Table III shows the correlations between parameters related to arteriosclerosis and GCF-Lf or GCF-AT; no significant correlation was observed for any of the parameters. The GCF-Lf level for 56 patients (77.8%) was < 0.4 µg/ mL; for 10 (13.9%) patients was within the range 0.40–0.79 µg/mL; and for 6 (8.3%) patients was ≥ 0.8 µg/mL. The GCFAT level for 62 (86.1%) patients was < 0.4 µg/mL; for 5 (6.9%) patients was within the range 0.40–0.79 µg/mL; and for 5 (6.9%) patients was ≥ 0.8 µg/mL. The max IMT, plaque score, baPWV, and FMD were compared on the basis of GCF-Lf and
GCF-AT levels; no significant differences were found between the 3 groups (Figure).
Discussion The principal finding of this study is that there were no significant correlations between any arteriosclerosis parameter (ie, max IMT, plaque score, baPWV, and FMD) and GCF-Lf or GCF-AT in patients with CAD. Moreover, no significant differences in GCF-Lf and GCF-AT levels were observed between patients with single, double, and triple-vessel CAD. Our study demonstrated the absence of a strong correlation between periodontal condition and parameters of arteriosclerosis status in patients with CAD. Only one report that examined the relationship between GCF biomarkers and atherosclerosis has been published.6) It reported that increased GCF concentrations of the inflammatory biomarker were significantly correlated with periodontitis and atherosclerosis. However, only carotid ultrasound examination was used to assess arteriosclerosis and the number of subjects was small. In our study, we examined a range of parameters for determining arteriosclerosis, and included a larger number of subjects than did the previous study. Unexpectedly, our results did not indicate any relationship between the GCF biomarkers and any of the parameters for atherosclerosis, including aortic stiffness and vascular endothelial function. Periodontal disease and atherosclerotic vascular disease share common risk factors such as smoking, diabetes mellitus, aging, and obesity.1,15) Because these represent confounding factors for periodontal disease and atherosclerotic vascular disease, it is difficult to obtain evidence regarding a correlation between these two diseases.1) It has been established that systemic inflammatory markers such as C-reactive protein and interleukin-6 are predictive markers for cardiovascular events.16-18) Periodontal disease is similarly associated with increased systemic inflammatory markers, including C-reactive protein and interleukin-6.18-20) Thus, the association between periodontal disease and atherosclerosis presented in previous reports 19,20) may not be a direct relationship; however, it may be a reflection of the increased levels of inflammatory markers in both diseases. Our results showing the nonexistence of a strong correlation between periodontal condition and arteriosclerosis severity were consistent with the recent American Heart Association statement.1) This study has several limitations. First, we did not have a control group of patients without CAD because it would be difficult to match the confounding factors discussed earlier between the CAD and the non-CAD groups. Second, the number of patients with abnormally high GCF-Lf and GCF-AT levels was small because we only included patients with at least 10 teeth to accurately collect GCF. Concentrations of biomarkers
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Int Heart J November 2015
Figure. Comparison of atherosclerosis parameters based on lactoferrin (GCF-Lf) and α1-antitrypsin (GCF-AT) levels in gingival crevicular fluid. A, B: Maximum intima–media thickness (max IMT). C, D: plaque score. E, F: brachial–ankle pulse wave velocity (baPWV). G, H: flow-mediated dilation (FMD). Data are presented as the mean ± standard deviation.
and the volume of GCF can differ among the local sites of different teeth according to the local periodontal inflammatory conditions. In the present study, we did not collect GCF samples from certain sites of teeth, but rather from 10 selected teeth from each subject; therefore, the method did not allow a full reflection of local inflammatory conditions. However, as we wished to evaluate the personal conditions rather than the regional conditions in the mouth, this method fulfilled our purpose. Third, this was a cross-sectional study that did not exam-
ine periodontal condition over time. Because oral care advice was not proactively given to our patients before collecting the GCF, we believe that casual GCF biomarkers levels reflected the average periodontal status. Finally, our data did not contain any parameters of periodontal disease severity other than GCFLf and GCF-AT, including pocket depth, clinical attachment level, and bleeding on probing;21) these should be addressed in future research. In conclusion, no correlation between the GCF biomark-
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ers and the severity of arteriosclerosis was detected. This result suggests that worsening of the periodontal condition as assessed by GCF biomarkers is not a major potential risk factor for arteriosclerosis.
10. 11.
Acknowledgment The GCF-Lf and GCF-AT measurements were performed at no cost thanks to the provision of Perio-catcher equipment from Ikagaku Company, Limited.
Disclosure
12.
Conflict of interests: Masataka Sata has received research funding from Takeda, Tanabe-Mitsubishi, Astellas, DaiichiSankyo, MSD, Bayer Healthcare, and Ono, and lecture fees from Takeda, Boehringer Ingelheim, Bayer Healthcare, Mochida, Astellas, Tanabe-Mitsubishi, Novartis, Astra-Zeneca, MSD, and Shionogi. 13.
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