Periodontitis, diabetes mellitus, cardiovascular disease: A Bermuda Triangle - Thesis

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Periodontitis, diabetes mellitus, cardiovascular disease

A Bermuda Triangle

Teeuw, W.J.

Publication date

2017

Document Version

Final published version

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Citation for published version (APA):

Teeuw, W. J. (2017). Periodontitis, diabetes mellitus, cardiovascular disease: A Bermuda

Triangle.

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Periodontitis,

Diabetes mellitus,

Cardiovascular disease

Wijnand J. Teeuw

Periodontitis,

Diabetes mellitus,

Cardiovascular disease

Wijnand J. Teeuw

A Bermuda Triangle

A Be rm ud a Triangle

voor het bijwonen

van de openbare

verdediging van

mijn proefschrift

op vrijdag 10 maart 2017

om 11.00 uur

in de Oude Lutherse kerk

Singel 411 te Amsterdam

na afloop van de promotie

bent u van harte welkom op

de receptie ter plaatse in de

Tetterode Bibliotheek

Wijnand Teeuw

W.Teeuw@acta.nl

Uitnodiging

Paranimfen

Wayne de Leeuw Rik van Vulpen promotie.teeuw@gmail.com

Periodontitis,

Diabetes mellitus,

Cardiovascular disease

A Bermuda Triangle

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Periodontitis,

Diabetes mellitus,

Cardiovascular disease

A Bermuda Triangle

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The studies described in this thesis were conducted at the Department of Periodontology of the Academic Centre for Dentistry Amsterdam (ACTA), the combined faculty of dentistry of the University of Amsterdam & VU University Amsterdam and at the Medical Center Slotervaart, Amsterdam.

Publication of the thesis and organization of the ceremony were generously supported by:

Cover design by Rik van Vulpen

Layout and printed by Gildeprint, Enschede, The Netherlands ISBN: 978-90-9030198-3

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Periodontitis,

Diabetes mellitus,

Cardiovascular disease

A Bermuda Triangle

ACADEMISCH PROEFSCHRIFT

ter verkrijging van de graad van doctor aan de Universiteit van Amsterdam

op gezag van de Rector Magnificus prof. dr. ir. K.I.J. Maex

ten overstaan van een door het College voor Promoties ingestelde commissie, in het openbaar te verdedigen in de Aula der Universiteit

op vrijdag 10 maart 2017, te 11.00 uur

door Wijnand Jan Teeuw geboren te Hoogblokland

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Promotiecommissie:

Promotor: Prof. dr. B.G. Loos Universiteit van Amsterdam

Copromotor: Dr. V.E.A. Gerdes Universiteit van Amsterdam

Overige leden: Prof. dr. F. Abbas Rijksuniversiteit Groningen

Prof. dr. J. de Lange Universiteit van Amsterdam

Prof. dr. M. Nieuwdorp Universiteit van Amsterdam

Prof. dr. F.R. Rozema Universiteit van Amsterdam

Prof. dr. U. van der Velden Universiteit van Amsterdam

Prof. dr. G.A. van der Weijden Universiteit van Amsterdam

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Chapter 1

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PERIODONTITIS

Periodontitis is a common chronic multifactorial inflammatory disease of the supporting structures of the teeth (root cementum, gingiva, periodontal ligament and alveolar bone) and is a major cause of tooth loss (Pihlstrom et al., 2005). The severe form of this condition occurs in about 10-15% of the population, and the prevalence is twice as high in subjects >50 years of age (Eke et al., 2012, Norderyd et al., 2015, Thornton-Evans et al., 2013). Two major clinical entities of periodontitis, the aggressive and chronic form, have been accepted (Armitage, 1999), but a clear distinction between these two phenotypes is difficult.

Overall, the current concept of the etiology of periodontitis is that it is a complex disease, where several causal factors play a role simultaneously. So far, five clusters of causal risk factors have been described: i) the environment, which is the subgingival microbiota, ii) individual (epi)genetic variations, iii) life style and behavior, iv) systemic diseases, and v) others (including iatrogenic causes, occlusal disturbances and unknown factors) (Loos et al., 2015). However, it is important to note that the relative contribution of each of the causal factors varies from patient to patient. In general, older patients with chronic periodontitis are considered to have a major contribution from environmental and lifestyle factors. So, many years of biofilm accumulation and unfavorable lifestyle behaviors like smoking, poor diet, and no or irregular visits to dental professionals, likely make substantial contributions to disease progression. On the other hand, periodontitis in younger patients, for example suffering from aggressive periodontitis, can be caused to a greater extent by genetic factors (Laine et al., 2012, Loos et al., 2015, Mucci et al., 2005, Stabholz et al., 2010). Although the relative contribution of several causal factors may be different among patients, the uncontrolled inflammatory response upon bacterial biofilm seems the major factor determining the onset and/or progression of the disease (Ling et al., 2015, Loos et al., 2015, Matthews et al., 2007, Preshaw and Taylor, 2011).

Besides the possibility that systemic diseases, such as diabetes mellitus (DM) (Preshaw et al., 2012), may contribute to the onset and/or progression of periodontitis, inflamed

periodontal tissueopens up the possibility for oral bacteria to enter the circulation and

induce systemic inflammation. It is now widely accepted that a low grade systemic inflammation contributes to metabolic dysregulation (King, 2008, Pradhan et al., 2001) and atherogenesis (Kaptoge et al., 2010, Friedewald et al., 2009). Indeed, periodontitis has been associated with DM (Preshaw et al., 2012, Lalla and Papapanou, 2011) and cardiovascular disease (CVD) (Dietrich et al., 2013, Lockhart et al., 2012, Schenkein and Loos, 2013).

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DIABETES MELLITUS

DM is a growing health problem and the prevalence is increasing dramatically (Lam and LeRoith, 2012). The global prevalence of DM in adult population has increased from 4.7% (108 million) in 1980 to 8.5% in 2014 (422 million). This indicates that the global prevalence (age-standardized) of DM has nearly doubled since 1980 until 2014 (World Health Organisation (WHO), 2016b), and this is expected to rise to 8.8% (592 million) by 2035 (Guariguata et al., 2014). The elevated blood glucose levels (severe or subtle) result in advanced glycation endproduct (AGE) formation in diabetes patients. AGEs have been shown to bind to specific receptors (RAGE) and induce hyperactivity of different cells, such as fibroblasts, endothelial cells and macrophages (Llambes et al., 2015). Therefore, AGEs produced by chronic hyperglycemia can cause an increase in endothelial dysfunction, hyper inflammatory responses, altered healing and increased predisposition to infections (Llambes et al., 2015, Muller et al., 2005). It has been suggested that these pathways, that are thought to lead to the classic microvascular and macrovascular complications of DM, are also involved in the pathophysiology of periodontitis (Llambes et al., 2015, Mealey and Rose, 2008).

DM and periodontitis are two chronic diseases that have long been considered to be biologically linked in a bi-directional way (Llambes et al., 2015, Preshaw et al., 2012, Lalla and Papapanou, 2011).

The diabetes mellitus їƉĞƌŝŽĚŽŶƚŝƚŝƐĐŽŶŶĞĐƚŝŽŶ

A large amount of case-reports, cross-sectional studies, longitudinal studies and reviews report the adverse effects of DM on the onset, progression and severity of periodontitis (Lalla and Papapanou, 2011, Llambes et al., 2015, Mealey and Rose, 2008, Taylor and Borgnakke, 2008). The prevalence of periodontitis in diabetes patients is estimated to be

two or even three times higher than in an otherwise healthy population

(Llambes et al.,

2015, Mealey et al., 2006)

. In addition, periodontitis is strongly associated with

uncontrolled DM (Eke et al., 2016, Garcia et al., 2015) due to the increased susceptibility for infections and impaired wound healing (Muller et al., 2005). Therefore, periodontitis is considered to be a complication of DM (Lalla and Papapanou, 2011, Löe, 1993, Preshaw et al., 2012) and it has been suggested that the dental setting might be suitable for diabetes screening (Lalla and Lamster, 2012).

dŚĞƉĞƌŝŽĚŽŶƚŝƚŝƐї diabetes mellitus ĐŽŶŶĞĐƚŝŽŶ

There is a growing body of evidence supporting the fact that the periodontal inflammation adversely affects glycemic control (Engebretson and Kocher, 2013, Preshaw et al., 2012). One biological explanation might be the increased pro-inflammatory state due to periodontitis (Schenkein and Loos, 2013), resulting in increased insulin resistance (King,

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2008, Shoelson et al., 2006). For example, Iwamoto et al. (2001) already suggested that periodontal treatment in type 2 diabetes patients is effective in reducing plasma levels of glycated hemoglobin (HbA1c), possibly through improved insulin resistance (Iwamoto et al., 2001). HbA1c reflects the plasma glucose levels over the past two or three months and ĂůĞǀĞůŽĨчϳй;ϱϯ ŵŵŽůͬŵŽůͿŝƐĐŽŶƐŝĚĞƌĞĚĂƐŵĞƚĂďŽůŝĐĂůůǇǁĞůů-controlled in diabetes patients (American Diabetes Association, 2015). Each reduction in HbA1c plasma levels is clinically relevant, since it will result in less diabetes complications (Genuth et al., 2003, Unger, 2008). Because periodontal treatment may improve glycemic control in diabetes patients, it may also contribute to the prevention and/or decrease in the progression of diabetes complications.

In general, standard diabetes care is focused on improving metabolic control, thereby reducing insulin resistance, and thus prevention of complications (American Diabetes Association, 2015, Rutten et al., 2013). In the Netherlands, diabetes patients are regularly checked preventively for common diabetes complications, especially retinopathy, neuropathy and nephropathy (Rutten et al., 2013). Because of the above mentioned bi-directional relationship between DM and periodontitis, implementation of oral care in diabetes care is desirable. In the Netherlands, oral care and diabetes care are well organized. However, a communication between the dentist and the diabetes care provider is nearly non-existent.

ATHEROSCLEROTIC CARDIOVASCULAR DISEASE

ACVD is a collective name for vascular pathologies in which the process of atherogenesis is the underlying etiologic mechanism. Atherogenesis results in endothelial dysfunction, arterial stiffness, reduction of blood vessel lumen size, high blood pressure, and,

ultimately, ischemic events (Patel et al., 2016).Atherosclerosis, and especially the rupture

of the atherosclerotic plaques, typically leads to symptoms of cardiovascular disease (CVD), such as angina, and CVD-related events, such as myocardial infarction and cerebrovascular accident (stroke) (Bentzon et al., 2014, Packard and Libby, 2008). CVD is the leading cause of death globally. An estimated 17.5 million people died from CVDs in 2012, representing 31% of all global deaths. Of these deaths, an estimated 7.4 million were due to coronary heart disease and 6.7 million were due to stroke (World Health Organisation (WHO), 2016a). Smoking, high blood pressure, elevated blood lipids, obesity and the presence of chronic diseases, like DM, rheumatoid arthritis, kidney disease etc. have been considered as typical risk factors for atherosclerosis (Herrington et al., 2016).

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Chronic metabolic or inflammatory diseases, such as DM, rheumatoid arthritis and periodontitis have been proposed to cause a pro-inflammatory state and may enhance atherogenesis (Kammoun et al., 2014, Kraakman et al., 2016, Schenkein and Loos, 2013). Indeed, epidemiological studies confirmed an independent association between periodontitis and ACVD (Lockhart et al., 2012). One of several plausible explanations for this association might be that complex diseases often share genetic risk factors

(Pleiotropy) (Loos et al., 2015, Vaithilingam et al., 2014). Recently, ANRIL, has been

associated with periodontitis (Schaefer et al., 2013, Schaefer et al., 2011). ANRIL is a genetic risk factor for several conditions with inflammatory components in Caucasians, and the strongest susceptibility locus for several types of ACVD, like coronary artery disease (CAD), myocardial infarction, abdominal aortic aneurysm and intracranial aneurysm (Deloukas et al., 2013, Helgadottir et al., 2008, Schunkert et al., 2008). Also, in a recent report, the CAD-associated gene WůĂƐŵŝŶŽŐĞŶ was significantly associated with the aggressive type of periodontitis (Schaefer et al., 2015). In conjunction with genetic risk factors, complex diseases share several negative lifestyle factors. For example, overweight and smoking have been proven to be major risk factors for CVD (Perk et al., 2012) and both factors are also associated with periodontal inflammation (Suvan et al., 2011, Warnakulasuriya et al., 2010).

Next to the overlapping genetic and lifestyle risk factors between ACVD and periodontitis, a direct effect of periodontitis on ACVD has been suggested and might explain the observed association between both diseases. In what way periodontitis might contribute to ACVD is not clear, but several causal mechanisms have been proposed whereby bacterial pathogens, antigens, endotoxins, and/or inflammatory cytokines from the periodontal lesions contribute to the process of atherogenesis and/or to thromboembolic events, thereby increasing the risk for ACVD (Lockhart et al., 2012, Schenkein and Loos, 2013, Tonetti et al., 2013). Periodontal therapy reduces periodontal inflammation and several treatment studies are highly suggestive that this reduction results in lower levels of markers of systemic inflammation (Bokhari et al., 2012, Sun et al., 2011) and improved vascular health (D'Aiuto et al., 2013).

C-reactive protein (CRP), an acute phase reactant, is a widely accepted key marker of atherosclerosis and is strongly associated with increased risk for ACVD; levels of 1-3 mg/L and >3 mg/L are considered to give medium and high risk for ACVD, respectively (Geluk et al., 2008, Ridker et al., 2010). On average periodontitis patients show elevated C-reactive protein levels between 1-3 mg/L, corresponding with medium risk for ACVD (Loos et al., 2000, Paraskevas et al., 2008). Furthermore, several studies indicated that periodontal treatment might reduce CRP-levels, suggesting that periodontitis causes an increased pro-inflammatory state and contributes to atherosclerosis and increased risk for ACVD. However, variations in levels have been shown both between patients and between

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1

populations (D'Aiuto et al., 2013, Paraskevas et al., 2008). Also the reduction of CRP levels after periodontal treatment varies substantially between study groups (D'Aiuto et al., 2013, Paraskevas et al., 2008), suggesting that other additional factors (genetic, lifestyle, etc.) may influence the systemic inflammatory response and consequently the risk for ACVD in patients suffering from periodontitis.

In addition to being a widely used biomarker for systemic inflammation and increased risk for ACVD, the actual functional role of CRP in periodontitis and maintenance of oral health is unknown. For more than two decades, it has been recognized that CRP functions as opsonin, which traditionally increases bacterial clearance (Du Clos and Mold, 2004, Tilg et al., 1993). This is accomplished by activation of the classical complement pathway, however it has also been shown that CRP could inhibit the alternative complement pathway by recruiting factor H (Holers, 2008, Mihlan et al., 2011, Mold et al., 1984, Suankratay et al., 1998), thus a modulating role for CRP can be suggested. Aberrant inflammatory responses to the dental biofilm have been suggested to be at the basis of the onset and/or progression of periodontitis (Ling et al., 2015, Loos et al., 2015, Matthews et al., 2007). In this perspective, the complement system is also suggested to play an important role in periodontal immune reactions (Hajishengallis et al., 2015). Therefore, CRP may play a role in maintaining oral health and in periodontitis by modulating the complement pathway.

AIMS OF THE THESIS

The outline of this thesis is depicted in Figure 1, representing the three diseases caught in a ‘Bermuda Triangle’. Because periodontitis is considered as an early complication of DM, it was hypothesized that the periodontal clinic could be a valuable location for diabetes screening. Since HbA1c levels were proposed as a suitable new marker for diabetes screening (American Diabetes Association, 2015) and a new validated chairside-method

for HbA1c testing was available, the hypothesis could be tested (Chapter 2). This study

explored whether periodontitis is an early sign of DM. Once there is periodontitis in patients with DM, it can be tested in what way periodontal therapy improves metabolic

control. In Chapter 3, the aim was to evaluate, by a systematic review and meta-analyses,

the available evidence regarding the effect of periodontal treatment on glycemic control in diabetes patients. Based on the results and subsequently the suggested necessity of implementation of oral care in diabetes care, a study was initiated to investigate the possibility for internists to obtain current oral health information from the dentist to

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Figure 1 dŚĞ ƌĞůĂƚŝŽŶƐŚŝƉ ďĞƚǁĞĞŶ ƉĞƌŝŽĚŽŶƚŝƚŝƐ͕ ĚŝĂďĞƚĞƐ ŵĞůůŝƚƵƐ ĂŶĚ ĂƚŚĞƌŽƐĐůĞƌŽƚŝĐ ĐĂƌĚŝŽǀĂƐĐƵůĂƌ ĚŝƐĞĂƐĞ͕

ĚĞƉŝĐƚĞĚŝŶŽƵƚůŝŶĞƐŽĨƚŚĞĞƌŵƵĚĂdƌŝĂŶŐůĞ͘dŚĞƐƚƵĚŝĞƐŝŶƚŚŝƐƚŚĞƐŝƐŵĂŝŶůǇĨŽĐƵƐŽŶƚŚĞƌĞůĂƚŝŽŶƐŚŝƉďĞƚǁĞĞŶ ƉĞƌŝŽĚŽŶƚŝƚŝƐĂŶĚĚŝĂďĞƚĞƐŵĞůůŝƚƵƐ;ďůĂĐŬ-ďůƵĞůŝŶĞͿĂŶĚƉĞƌŝŽĚŽŶƚŝƚŝƐĂŶĚĂƚŚĞƌŽƐĐůĞƌŽƚŝĐĐĂƌĚŝŽǀĂƐĐƵůĂƌĚŝƐĞĂƐĞ ;ďůĂĐŬ-ƌĞĚ ůŝŶĞͿ͘ /Ŷ ƚŚŝƐ ƉĞƌƐƉĞĐƚŝǀĞ͕ ƐŽŵĞ ƐƚƵĚŝĞƐ ĂůƐŽ ĚŝƐĐƵƐƐ ƚŚĞ ĐŽŶƐĞƋƵĞŶĐĞ ŽĨ ƚŚĞ ĞǆŝƐƚŝŶŐ ƌĞůĂƚŝŽŶƐŚŝƉ ďĞƚǁĞĞŶĚŝĂďĞƚĞƐŵĞůůŝƚƵƐĂŶĚĂƚŚĞƌŽƐĐůĞƌŽƚŝĐĐĂƌĚŝŽǀĂƐĐƵůĂƌĚŝƐĞĂƐĞ;ůŝŐŚƚĐŽůŽƌĞĚďůƵĞ-ƌĞĚůŝŶĞͿ

To achieve more understanding of the association between periodontitis and ACVD, a study was performed to explore whether genetic variation could explain to some extent the observed increased systemic inflammation in periodontitis patients. In this study, CRP

was used as a biomarker for systemic inflammation (Chapter 5). In addition, a study was

initiated to investigate the possible relationship between subclinical atherosclerosis and periodontitis, using a recently identified clinical risk marker for atherosclerosis and ACVD (Chapter 6). Based on the observed association between periodontitis and ACVD, it was

hypothesized that periodontal treatment could contribute to vascular health. In Chapter

7, the aim was to evaluate, by a systematic review and meta-analyses, the available

evidence whether periodontal treatment contributes to vascular health, by improving the atherosclerotic profile in periodontitis patients with and without comorbidity. The last

study in this thesis (Chapter 8) is an explorative study and was performed to investigate

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systemic inflammation and increased risk for ACVD, the role of CRP in periodontitis is unknown. It has been demonstrated that CRP can modulate the complement system and may therefore play a role oral health. As a first step, the presence of CRP and other complement factors in the oral cavity was determined, and the results may direct further research.

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1

Chapter 2

Periodontitis as a possible early sign of

diabetes mellitus

Wijnand J. Teeuw, Madeline X.F. Kosho, Dennis C.W. Poland,

Victor E.A. Gerdes, Bruno G. Loos

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ABSTRACT

Objective The early diagnosis of (pre)diabetes mellitus is essential for the prevention of

diabetes complications. It has been suggested that gum disease (periodontitis) might be an early complication of diabetes and may be a useful risk indicator for diabetes screening. Therefore, a dental office could be a good location for screening for (pre)diabetes in periodontitis patients, using a validated hemoglobin A1c (HbA1c) dry spot analysis.

Research Design and Methods A total of 313 individuals from a university dental clinic

participated. From 126 mild/moderate periodontitis patients, 78 severe periodontitis patients and 109 subjects without periodontitis, HbA1c values were obtained by the analysis of dry blood spots. Differences in mean HbA1c values and the prevalence of (pre)diabetes between the groups were analyzed.

Results The mild/moderate and severe periodontitis groups showed significantly higher

HbA1c values (6.1% ± 1.4% [43 mmol/mol ± 15 mmol/mol] and 6.3% ± 1.3% [45 mmol/mol ± 15 mmol/mol], respectively) compared with the control group (5.7% ± 0.7% [39 mmol/mol ± 8 mmol/mol], p=0.003). In addition, according to the ADA guidelines for diagnosis, there was a significant overrepresentation of subjects with suspected diabetes (23% and 14%) and prediabetes (47% and 46%) in the severe periodontitis group and mild/moderate periodontitis groups, respectively, compared to the control group (10% and 37%, p=0.010). Notably, 18.1% of suspected new diabetes patients were found among subjects with severe periodontitis compared to 9.9% and 8.5% among subjects with mild/moderate periodontitis and controls, respectively (p=0.024).

Conclusions The dental office, with particular focus on severe periodontitis patients,

proved to be a suitable location for screening for (pre)diabetes; a considerable number of suspected new diabetes cases were identified. The early diagnosis and treatment of (pre)diabetes help to prevent more severe complications and benefit the treatment of periodontitis.

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2

INTRODUCTION

Diabetes mellitus is a growing health problem and its prevalence is dramatically increasing (Lam and LeRoith, 2012). The prevalence of diabetes was estimated at 285 million adults worldwide in 2010 (Shaw et al., 2010), and this is expected to rise to 552 million by 2030 (Whiting et al., 2011). However, due to the absence of symptoms and/or disease-related knowledge, diabetes often goes undetected, and approximately one-third of people with diabetes are not aware of their status (Cowie et al., 2006, Kim et al., 2006). The early diagnosis and intervention of (pre)diabetes prevent the common micro- and macro-vascular complications (Tian et al., 2014) and are cost-effective (Herman et al., 2005). Therefore, risk indicators for (pre)diabetes screening are needed and proposed (American Diabetes Association, 2015). In this respect, the onset of several oral pathologies might be indicative of metabolic dysregulation.

Several studies demonstrate the association between diabetes and oral diseases. The most commonly observed chronic oral disease is periodontitis. Periodontitis is a common chronic multifactorial inflammatory disease of the supporting structures of the teeth (root cementum, gingiva, periodontal ligament and alveolar bone), initiated and sustained by an aberrant host immune response against resident bacterial biofilm on the teeth. It is a major cause of tooth loss (Pihlstrom et al., 2005). The severe form of this condition occurs in approximately 10-15% of the population, and the prevalence is twice as high in subjects >50 years of age (Burt et al., 2005, Eke et al., 2012b, Hugoson and Norderyd, 2008). Many studies have demonstrated that diabetes mellitus severely exacerbates the onset, progression and severity of periodontitis (Demmer et al., 2012, Lalla and Papapanou, 2011, Mealey and Rose, 2008, Taylor and Borgnakke, 2008). The prevalence of periodontitis in diabetes patients is estimated to be two or even three times higher than in an otherwise healthy population(Mealey et al., 2006). In addition, periodontitis is strongly associated with uncontrolled diabetes mellitus (Garcia et al., 2015, Eke et al., 2016). This is because diabetic subjects, particularly uncontrolled diabetic subjects, are more susceptible to infections and impaired wound healing (Muller et al., 2005); therefore, periodontitis is considered to be a complication of diabetes mellitus (Lalla and Papapanou, 2011, Löe, 1993, Preshaw et al., 2012). With this knowledge, it has been suggested that dentists could help screen for (pre)diabetes (Lalla et al., 2011, Strauss et al., 2010). The majority of dental practices are not equipped for blood biochemistry. The measurements of hemoglobin A1c (HbA1c) using dry blood spots may be a conservative way to screen for diabetes in patients with periodontitis.

The aim of the present study was to determine HbA1c levels and affirm the presence of (pre)diabetes in subjects from a university dental clinic with and without periodontitis using the analysis of dry blood spots. Because periodontitis can be considered a

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complication of (pre)diabetes, we suggest the hypothesis that periodontitis patients show an increased prevalence of (pre)diabetes compared with subjects without periodontal disease.

RESEARCH DESIGN AND METHODS Study Population

A consecutive series of periodontitis patients who were referred to the Department of Periodontology of the Academic Centre for Dentistry Amsterdam (ACTA) for the diagnosis and treatment of periodontitis were enrolled in this study. Controls were selected among subjects registered for restorative dental procedures or who visited the dental school for regular dental checkups. The selection period was between February 2014 and September 2015. A total of 313 subjects, including 78 patients with severe periodontitis, 126 patients with mild/moderate periodontitis and 109 controls, were included in this study. Briefly, referred periodontitis patients were recruited during their first visit to the periodontal clinic. Probing pocket depth (PPD) measurements and gingival recession (GR), if present, were recorded at six sites per tooth and dental radiographs were available to analyze interproximal alveolar bone levels. For those sites where the GR was recorded, the AL could be determined (PPD+GR). Patients were classified as suffering from periodontitis using the CDC-AAP case definition (Eke et al., 2012a): mild/moderate was defined as having at least 2 inƚĞƌƉƌŽǆŝŵĂůƐŝƚĞƐǁŝƚŚ>шϯŵŵ and at least 2 interproximal sites with WWшϰŵŵ;ŶŽƚŽŶƐĂŵĞƚŽŽƚŚͿŽƌŽŶĞƐŝƚĞǁŝƚŚWWшϱŵŵ(Eke et al., 2012a). Patients who sŚŽǁĞĚĂƚůĞĂƐƚϮŝŶƚĞƌƉƌŽǆŝŵĂůƐŝƚĞƐǁŝƚŚ>шϲŵŵŽŶĚŝĨĨĞƌĞŶƚƚĞĞƚŚŝŶĐŽŶũƵŶĐƚŝŽŶ with at least 1 interproximal site with PPшϱŵŵ were classified as suffering from severe periodontitis (Eke et al., 2012a). All patients were untreated and showed generalized bleeding on probing. Control subjects were included if they did not fulfill any of the above mentioned criteria for case definition. In addition, these subjects showed no interproximal alveolar bone loss ŽŶчϭ-year-old dental bitewing radiographs. Subjects with an age of <18 years were excluded from the study. No other exclusion criteria were used.

All participants received verbal and written information about the purpose of the study and provided informed consent. The Medical Ethical Committee of the VU University Medical Center approved the study.

Background and Diabetes-related Characteristics

For all participants, general and diabetes-related characteristics were recorded with the help of a questionnaire and medical history (Hx). The recorded characteristics were from the questionnaire: sex, age, ethnicity (origin of parents), education level, smoking habits, relatives with diabetes mellitus and history of periodontal treatment; from the Hx:

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2

diagnosis of hypertension, hypercholesterolemia and diabetes mellitus. Height and weight were measured in our clinic to determine the body mass index (BMI).

Blood Collection and HbA1c Analysis

After a finger stick, two drops of capillary blood were spotted on a Whatman 903 paper collection card (GE Healthcare Life Sciences, Eindhoven, Netherlands). The collection cards were stored at 4°C and sent weekly by regular postal mail to the Clinical Chemistry Laboratory of the Medical Center (MC) Slotervaart, Amsterdam, the Netherlands. Two 6-mm punches were incubated in 400 PL hemolysis buffer of the standard HbA1c test, which was measured immunoturbidimetrically on an Abbott ci8200 clinical chemistry analyzer (Abbott Laboratories, Abbott Park, IL, USA) (Elefano et al., 2006). The finger stick and Whatman paper collection method was previously validated against the conventional EDTA-blood collection by venipuncture according to the Clinical and Laboratory Standards Institute (CLSI) EP9 method comparison protocol. Precision and stability studies of HbA1c on Whatman 903 collection cards have been reported (Fokkema et al., 2009) and showed that the finger stick collection card and punch method for HbA1c determination was highly correlated with the standard EDTA blood collection method (Regression analysis r=0.99; Supplementary Figure 1). To further determine the validity of the finger stick method in the current study population, 10 patients were selected for both the finger stick analysis and the standard EDTA venous blood collection method. A similar correlation in HbA1c between these two methods was obtained (regression analysis r=0.99, data not shown)

Statistical Analyses

Sample size was determined based on a pilot study, including 49 periodontally healthy subjects and 33 periodontitis patients. We detected a mean difference in the HbA1c of 0.7% (7 mmol/mol) between periodontitis patients and control subjects, which we considered clinically relevant. The calculation of sample size (http://powerandsamplesize .com) was based on a two-sided Student’s t-test of 5% with 80% power and using the mean of HbA1c of both groups (control: 5.5% [37 mmol/mol] and periodontitis: 6.2% [44 mmol/mol]) and an overall study population-based standard deviation of 1.3% (15 mmol/mol). The required sample size was a minimum of 61 subjects per group.

Data were analyzed with SPSS 23.0.0.2 (IBM SPSS, Chicago, IL, USA). The means, standard deviations, medians, total range and frequency distributions were calculated. According to the ADA guidelines (American Diabetes Association, 2015), HbA1c values were classified as normal (<5.7% [<39 mmol/mol]), pre-diabetes (5.7-6.4% [39-47 mmol/mol]) or diabetes mellitus ;шϲ͘ϱй΀шϰϴŵŵŽůͬŵŽů΁Ϳ. The general and diabetes-related characteristics of the study population, HbA1c levels, and (pre)diabetes prevalence within the study population

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were compared with parametric and non-parametric tests (ANOVA and chi-squared test). Where applicable, analyses were corrected for multiple testing (Bonferroni). For all analyses, the significance level was set to p<0.05.

RESULTS Study Population

In this study, a total of 313 consecutive subjects (109 controls, 126 mild/moderate and 78 severe periodontitis patients) were included. Demographic and diabetes-related characteristics of the study population are presented in Table 1. The mean ages for the control, mild/moderate and severe periodontitis group were 48.4, 51.1 and 50.5 years, respectively (P = 0.200). Overall differences in sex distribution between the groups were significant (P = 0.010), with more males in the severe group (62% compared to 40% in the mild/moderate periodontitis group and 47% in the control group). In addition, more subjects from the control group were in the highest education category compared to the periodontitis groups (P = 0.007). Among periodontitis patients, there were more current smokers (mild/moderate periodontitis: 23.0% and severe periodontitis: 37.2%), while this rate was 9.2% among controls (P < 0.001).

The BMI was significantly ŚŝŐŚĞƌ ŝŶ ƉĞƌŝŽĚŽŶƚŝƚŝƐ ƉĂƚŝĞŶƚƐ ;шϮϳ ŬŐͬŵ2_{) than in controls}

(24.9 kg/m2_{), and more patients in the periodontitis group could be classified as being}

overweight ;D/ шϮϱ ŬŐͬŵ2_{; P = 0.008). No significant differences could be observed}

between the three groups regarding the number of subjects reporting hypertension and hypercholesterolemia, while more mild/moderate periodontitis patients reported relatives with diabetes mellitus. The number of individuals with self-reported diabetes mellitus was not significantly different between the controls and periodontitis patients (2.8% for controls, 4.0% for mild/moderate periodontitis and 7.7% for severe periodontitis, P = 0.256, Table 2).

Periodontitis patients had significantly fewer teeth than controls (P < 0.001). The severe periodontitis group had more ƚĞĞƚŚǁŝƚŚшϱϬйĂůǀĞŽůĂƌďŽŶĞůŽƐƐ than the mild/moderate periodontitis group (median: 8 and 1, respectively). Controls had no alveolar bone loss.

HbA1c Analysis

The mean HbA1c values for the study groups are presented in Table 2. With an increasing severity of periodontitis, a significant increase of HbA1c levels could be observed (P = 0.003). On average, the mean HbA1c in the severe periodontitis group was 6.3% (45 mmol/mol), in the mild/moderate periodontitis group 6.1% (43 mmol/mol), and in the controls 5.7% (39 mmol/mol). Subsequently, we tabulated the number of subjects with suspected diabetes and prediabetes based on levels of HbA1c using the ADA guidelines

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(American Diabetes Association, 2015). There was a significant overrepresentation of subjects with suspected diabetes (23% and 14%) and prediabetes (47% and 46%) in the severe periodontitis group and mild/moderate periodontitis groups, respectively, compared to the control group (10% and 37%, Table 2).

Table 1 Background characteristics

Control (n = 109) Mild/moderate periodontitis (n = 126) Severe periodontitis (n = 78) P value Age (years) 48.4 ± 14.8 51.1 ± 11.5 50.5 ± 10.1 0.200 Sex, male (%) 51 (46.8%) 50 (39.7%) 48 (61.5%) 0.010 Origin, European (%) 84 (77.1%) 94 (74.6%) 53 (67.9%) 0.364 Education (%) 0.007 Low 13 (11.9%) 28 (22.2%) 21 (26.9%) Medium 30 (27.5%) 47 (37.3%) 27 (34.6%) High 66 (60.6%) 51 (40.5%) 30 (38.5%) BMI (kg/m2₎ _{24.9 ± 3.7} _{27.2 ± 4.6}† _{27.0 ± 4.7}† _0.000 D/шϮϱŬŐͬŵ2_(%) _{48 (44.0%)} _{80 (63.5%)} _{47 (60.3%)} _0.008 Smoking status (%) 0.000 Never 62 (56.9%) 34 (27.0%) 12 (15.4%) Former 37 (33.9%) 63 (50.0%) 37 (47.4%) Current 10 (9.2%) 29 (23.0%) 29 (37.2%) Hypertension (%)* _{23 (21.1%)} _{24 (19.0%)} _{14 (17.9%)} _0.855 Hypercholesterolemia* _{16 (14.7%)} _{22 (17.5%)} _{17 (21.8%)} _0.451 First-degree relative with

diabetes (%) 40 (36.7%) 71 (56.3%) 28 (35.9%) 0.002 # Teeth 28 (20-32) 27 (16-32)† _{26 (15-32)}†‡ _0.000 # Teeth ǁŝƚŚшϱϬйďŽŶĞůŽƐƐ NA 1 (0-6)§ _{8 (0-23)}|| _0.000 ηdĞĞƚŚǁŝƚŚWWшϲŵŵ NA 4 (0-22) 16 (1-32) 0.000 η^ŝƚĞƐǁŝƚŚWWшϲŵŵ NA 7.5 (0-71) 44.5 (1-115) 0.000 Data are presented as the mean ± SD, median and range, or as n (%). BMI, body mass index. PPD, probing pocket depth.

*_{Based on the medical history (Hx)}

†_{Bonferroni test: P-value <0.05 compared with control}

‡_{Bonferroni test: P-value <0.05 compared with mild/moderate periodontitis} §_{n = 122}

||_{n = 72}

The ADA HbA1c cut-off for diabetes (6.5%, [48 mmol/mol]) might explain the relatively high prevalence of suspected diabetes in the control group (10%) compared to the estimated overall prevalence of diabetes in the Netherlands (5%) (Kleefstra et al., 2016). Because a threshold of HbA1c of 7% (53 mmol/mol) has been proposed to exclude possible false positive measurements (Saudek et al., 2008, van 't Riet et al., 2010), the

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prevalence of study subjects with HbA1c ш7% ;шϱϯŵŵŽůͬŵŽůͿ was determined: control, 3.7%; mild/moderate periodontitis, 7.1% and severe periodontitis, 12.8% (P = 0.061).

Table 2 HbA1c values and the suspected diagnosis of (pre)diabetes*_{with and without patients with diabetes} based on medical history

Control (n=109) Mild/moderate periodontitis (n=126) Severe periodontitis (n=78) P-value Diabetes, Hx (%)† _{3 (2.8%)} _{5 (4.0%)} _{6 (7.7%)} _0.256 HbA1c (%) (mmol/mol) All subjects‡ _{5.7 ± 0.7 (39 ± 8)} _{6.1 ± 1.4 (43 ± 15)} _{6.3 ± 1.3}¶_{(45 ± 15)} _0.003 Subjects without diabetes§|| 5.7 ± 0.6 (38 ± 7) 6.0 ± 1.3 (42 ± 15) 6.2 ± 1.2 ¶_{(44 ± 13)} _0.009 # Suspected diagnosis (%) All subjects‡ _0.010 Healthy 58 (53.2%) 51 (40.5%) 23 (29.5%) Prediabetes 40 (36.7%) 58 (46.0%) 37 (47.4%) Diabetes 11 (10.1%) 17 (13.5%) 18 (23.1%) Subjects without diabetes§|| _0.024 Healthy 58 (54.7%) 51 (42.1%) 23 (31.9%) Prediabetes 39 (36.8%) 58 (47.9%) 36 (50.0%) Diabetes 9 (8.5%) 12 (9.9%) 13 (18.1%) Data are presented as the mean ± SD or as n (%)

*_{(pre)diabetes classification according to the ADA guidelines 2015} † _{Based on the medical history (Hx)}

‡_{Subjects with and without diabetes based on the medical history are included.} §_{Subjects with diabetes based on the medical history are excluded}

|| _{Control: n=106; Mild/moderate periodontitis: n=121; Severe periodontitis: n=72} ¶_{Bonferroni test: P-value <0.05 compared with control}

To further explore the potential role of periodontitis as a risk indicator for diabetes and its contribution to diabetes screening, we determined the percentage of patients without a Hx of diabetes that ŚĂĚ Ă ,ďϭĐ шϲ͘ϱй ;шϰϴ mmol/mol, new diabetes). In all groups, suspected new diabetes patients were found, and this number increased with increasing severity of periodontitis (control: 8.5%, mild/moderate periodontitis: 9.9% and severe periodontitis: 18.1%, P = 0.024, Table 2).

CONCLUSIONS

In this study, we aimed to evaluate HbA1c values in subjects without and with periodontitis to establish the prevalence of (pre)diabetes, as periodontitis has been considered a complication of (pre)diabetes (Lalla and Papapanou, 2011). This study used a validated finger stick test for HbA1c with the advantage of a chairside test and the

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accuracy of the standard immunoturbidimetrical assessment of HbA1c on a clinical analyzer applying the new ADA guidelines for the diagnosis of diabetes (American Diabetes Association, 2015). Therefore, this method is very useful for the screening of (pre)diabetes in a dental office setting. The idea of diabetes screening in the dental office has been suggested by several other research groups, although their reports may have had different aims and techniques among themselves (Lalla et al., 2015, Lalla et al., 2013, Lalla et al., 2011, Rosedale et al., 2016, Strauss et al., 2015). The early identification of subjects at high risk for (pre)diabetes mellitus or with undiagnosed diabetes mellitus is crucial to implement measures that may prevent or delay progression from prediabetes to overt diabetes mellitus and reduce the incidence of chronic complications (Bianchi et al., 2012). For example, the results of a 20-year follow-up study showed that early prevention strategies, such as lifestyle intervention, resulted in a reduction in diabetes mellitus onset and consequently, it was associated with a 47% reduction in the incidence of threatening retinopathy (Gong et al., 2011). Recently, a 23-year follow-up of the latter study showed that early lifestyle intervention was associated with a decrease in the cumulative incidence of cardiovascular and all-cause mortality (Li et al., 2014). Current ADA recommendations for the management of (pre)diabetes reflect these observations and risk indicators for (pre)diabetes screening are needed and proposed (American Diabetes Association, 2015). Notably, among subjects with severe periodontitis, we found more than 18% of the subjects who were unaware of the presence of diabetes had HbA1c values exceeding the 6.5% (48 mmol/mol) threshold. This confirms the assumption that severe periodontitis could be an early sign of undiagnosed diabetes. In this respect, the presence of severe periodontitis might be indicative of metabolic dysregulation and by the identification of diabetes, treatment of this disease can be initiated, which is also relevant for the successful treatment of periodontitis. We expect that a good collaboration between dentists and diabetologists will lead to the best treatment results.

One recent study used a point-of-care (POC) HbA1c assessment by means of a finger stick blood sample but employed a benchtop analyzer (Holm et al., 2016). Although this test obtains rapid results, the accuracy compared to values obtained by the standard assessment of HbA1c is questionable (Manley et al., 2014). In contrast, our finger stick and Whatman paper collection method and the standard immunoturbidimetrical assessment of HbA1c on an Abbott ci8200 clinical chemistry analyzer (Elefano et al., 2006) showed a very high correlation with the venipuncture collection method and subsequent HbA1c measurement (r=0.99). Despite a less accurate HbA1c determination, the results of the recent study (Holm et al., 2016) corroborate our findings that a substantial number of suspected new diabetes patients in the periodontitis group can be found. A higher frequency of suspected new diabetes patients in the periodontitis group in our study can be explained by the higher accuracy of our method for HbA1c determination and a clear

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case definition for subjects without or with mild/moderate or severe periodontitis. Notably, subjects with severe periodontitis showed a high frequency of suspected new diabetes cases, and the applied ADA case definition may therefore be very useful in general practice. In our study, compared to the recent publication (Holm et al., 2016), the control and periodontitis groups were comparable regarding age and ethnic origin; these factors have been considered important for diabetes screening (American Diabetes Association, 2015).

The prevalence of suspected diabetes in the control group (10.1%) in the present study, as measured by HbA1c, is high compared to the overall estimated prevalence in the Netherlands (5.45%) (Kleefstra et al., 2015). An obvious explanation could be that our study population, derived from a dental school, is not representative of the Dutch population. Another reason might be that the national prevalence is actually an underestimation; because diabetes is often asymptomatic in its early stages, it may remain undiagnosed for many years (Harris et al., 1992). In addition, it is possible that the current use of HbA1c levels results in a higher prevalence compared to the prevalence based on the traditional parameter plasma glucose levels, such as fasting plasma glucose levels and/or the oral glucose tolerance test. The recently adopted diagnosis of diabetes by HbA1c has been proposed by the ADA (American Diabetes Association, 2015), and the advantages and disadvantages for HbA1c as a diagnostic marker has been extensively discussed in the literature (Carson et al., 2010, Christensen et al., 2010, Kramer et al., 2010, Lorenzo and Haffner, 2010, van 't Riet et al., 2010, Zhou et al., 2010). However, most of the studies show that even the use of HbA1c levels may result in an underestimation of diabetes prevalence. Nevertheless, a higher cut-off of HbA1c of ш7.0% ;шϱϯ mmol/mol, ŝŶƐƚĞĂĚŽĨшϲ͘ϱй ΀шϰϴŵŵŽůͬŵŽů΁) has been proposed to exclude a possible false positive diagnosis (Saudek et al., 2008, van 't Riet et al., 2010). When applying this threshold, the ƉƌĞǀĂůĞŶĐĞŽĨƐƵďũĞĐƚƐǁŝƚŚ,ďϭĐůĞǀĞůƐŽĨшϳ͘Ϭй ;шϱϯŵŵŽůͬŵŽůͿ in the control group was 3.7%, and this can be considered in agreement with the overall estimated prevalence of diabetes in the Netherlands. NeverthelĞƐƐ͕ĂƉƉůǇŝŶŐƚŚĞƚŚƌĞƐŚŽůĚшϳй ;шϱϯŵŵŽůͬŵŽůͿ, the prevalence of subjects with diabetes was clearly higher in those with mild/moderate periodontitis (7.1%) and with severe periodontitis (12.8%). Interestingly, the results show that half of the subjects classified as having diabetes͕ ƵƐŝŶŐ Ă ƚŚƌĞƐŚŽůĚ ŽĨ шϲ͘ϱй ;шϰϴ mmol/mol), do have HbA1c levels in the range of 6.5-7% (48-53 mmol/mol), confirming that periodontitis is a useful risk indicator for the screening of early diabetes. The early diagnosis and intervention of (pre)diabetes prevent the common micro- and macro-vascular complications (Tian et al., 2014) and are cost-effective (Herman et al., 2005). In addition, the early diagnosis and treatment of (pre)diabetes may also benefit the treatment of periodontitis.

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A dental office that treats severe periodontitis patients, is a suitable location for screening for (pre)diabetes; a considerable number of suspected new diabetes cases were identified and indicated that periodontitis is an early sign of diabetes mellitus.

ACKNOWLEDGEMENTS Funding

This research was supported in part by a grant of the University of Amsterdam for the focal point “Oral Infection and Inflammation”.

Conflicts of interest

The authors declare no conflicts of interest.

Authors’ Contributions

W.J.T. and B.G.L designed the study, analyzed the data and wrote the manuscript. M.X.F.K. designed the study, analyzed the data and contributed to the discussion. D.C.W.P. and V.E.A.G. designed the study, contributed to the discussion and reviewed/edited the manuscript.

Periodontitis, diabetes mellitus, cardiovascular disease: A Bermuda Triangle - Thesis

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