Research article

Lipopolysaccharide associates with pro-atherogenic 14(4) (2008) 247–253

© SAGE Publications 2008
lipoproteins in periodontitis patients Los Angeles, London,
New Delhi and Singapore
ISSN 1753-4259 (print)
K.A. Elisa Kallio1, Kåre Buhlin2, Matti Jauhiainen3, Ritva Keva1, 10.1177/1753425908095130

Anita M. Tuomainen1, Björn Klinge2, Anders Gustafsson2, Pirkko J. Pussinen2

1
Institute of Dentistry, University of Helsinki, and Department of Oral and Maxillofacial Diseases, Helsinki
University Central Hospital, Helsinki, Finland
2
Division of Periodontology, Institute of Odontology, Karolinska Institutet, Huddinge, Sweden
3
Department of Molecular Medicine, National Public Health Institute, Helsinki, Finland

Introduction: Periodontitis patients are known to suffer from endotoxemia, which may be among the major risk
factors for atherosclerosis. In health, lipopolysaccharide (LPS) is mainly carried with high density lipoprotein (HDL)
particles. Shift of LPS toward lipoproteins with lower densities may result in less effective endotoxin scavenging. Our
aim was to determine plasma LPS activity and lipoprotein-distribution before and after treatment in periodontitis
patients.
Patients and Methods: Very low and intermediate density (VLDL-IDL), low density (LDL), HDL2, HDL3, and
lipoprotein-deficient plasma (LPDP) were isolated by sequential ultracentrifugation. Patients included 34 subjects
aged 53.5 ± 8.3 years, before and 6 months after periodontal treatment.
Results: The mean LPS distribution decreased among lipoprotein classes as follows: VLDL-IDL 41.3 ± 12.1%, LPDP
25.0 ± 7.0%, HDL3 13.1 ± 5.2%, LDL 11.5 ± 3.7%, and HDL2 9.2 ± 2.8%. Plasma and VLDL-IDL-associated LPS
correlated positively, and LDL- and HDL-associated LPS negatively with clinical periodontal parameters and plasma
cytokine concentrations. Mean plasma LPS activity increased after periodontal treatment from 44.0 ± 17.0 to 55.7 ±
24.2 EU/ml (P = 0.006). No significant changes were found in LPS lipoprotein distribution and lipoprotein
compositions after the treatment.
Conclusions: Endotoxemia increases with severity of periodontitis. In periodontitis, LPS associates preferentially
with the pro-atherogenic VLDL-IDL fraction. Periodontal treatment has only minor effects on plasma LPS activity or
distribution, which reflects persistence of the disease.

Keywords: Infection, inflammation, lipoproteins, lipoprotein subclasses, periodontal disease, teeth

INTRODUCTION tions in adults. According to the Health-2000 survey

Periodontitis is a bacterial infection in tooth-supporting
tissues, which eventually leads to loss of teeth if left
untreated. Host response to bacterial insult leads to
chronic inflammation characterized by gingival bleed-
ing, formation of deepened periodontal pockets, and
destruction of periodontal ligaments and alveolar bone.
Periodontitis is one of the most common chronic infec-
conducted in Finland, 64% of adults had deepened peri-
odontal pockets and 21% severe periodontitis.1
Furthermore, 88% of Finnish adults harbour one or more
of the main periodontal pathogens in their saliva.2 When
periodontitis develops and progresses, the bacterial com-
position of the subgingival biofilm changes from the
dominance of Gram-positive bacteria to a majority
Gram-negative bacteria. Bacteria and their components

Received 24 April 2008; Revised 2 June 2008; Accepted 9 June 2008

Correspondence to: Elisa Kallio, Institute of Dentistry, University of Helsinki, PO Box 63, FI-00014 Helsinki, Finland
Tel: +358 9 19 125570; Fax. +358 9 19125194; E-mail elisa.kallio@helsinki.fi
248 Kallio, Buhlin, Jauhiainen et al.
may have direct access to circulation through bleeding
periodontal pockets. As a consequence, periodontitis
patients suffer from endotoxemia.3–5
About 80–97% of lipopolysaccharide (LPS) is bound
to lipoproteins in the circulation,6,7 and all main lipopro-
tein classes are involved.8 In the serum of healthy
donors, LPS preferentially binds to high-density lipopro-
teins (HDL), which promotes LPS neutralization.8 In
sepsis patients, however, the majority of LPS is bound to
very low-density lipoproteins (VLDL).9 Binding of LPS
to VLDL increases the hepatic uptake of the lipoprotein
3-fold, which in sepsis patients protects against LPS-
induced toxicity.10 LPS complexed with low-density
lipoprotein (LDL) or VLDL is also taken up by
macrophages in a process which gives rise to foam cell
formation and progression of atherosclerosis.11,12 LPS
activity in the circulation is determined by the levels of
its carrier proteins, i.e. all lipoprotein classes, as well as
lipopolysaccharide binding protein (LBP), CD14, and
phospholipid transfer protein (PLTP).13 Nevertheless, lit-
tle is known of the long-term effects of endotoxemia
associated with periodontitis.
Clinical periodontitis has been reported to be associ-
ated with an increased risk for cardiovascular diseases or
atherosclerosis,14,15 but contrary studies have also been
published.16 Several studies imply that periodontal
pathogens play an assisting role in atherogenesis: the
number of periopathogenic species,17 periodontal
pathogen burden,18 and seropositivity to the pathogens19
increase the risk for atherosclerosis in humans, and
administration of Porphyromonas gingivalis or
Aggregatibacter actinomycetemcomitans induce early
atherosclerotic alterations in animals.20–23 Since endotox-
emia is an independent risk factor for cardiovascular dis-
ease,24 it is justified to hypothesize that LPS contributes
to periodontitis-induced atherosclerosis.
The aim of this study was to determine plasma LPS
activity and lipoprotein distribution in periodontitis patients
before and after periodontal treatment. Also, the mass com-
positions of lipoprotein subclasses were determined.
PATIENTS AND METHODS
Patients and periodontal treatment
Thirty-four patients with periodontitis were included in
the study. Their mean age was 53.3 ± 8.1 years and body
mass index 26.4 ± 3.4 kg/m2. From the patients, 19 were
males and 15 females. Twenty patients did not smoke, 3
were former smokers, and 11 current smokers. All of the
smokers had been smoking for 10 years or more.
All patients had a minimum of seven sites exhibiting
at least 6 mm loss of clinical attachment. All had been
referred to a specialist clinic in periodontology due to
their severe periodontitis and were undergoing treatment
for the condition. All the patients had periodontitis
gravis et complicata25 in which there is a horizontal loss
of supporting tissue by one-third or more of the root-
length with bleeding on probing, furcation involvements
of the multirooted teeth and/or angular bony defects with
pus. Patients were excluded from the study if they had a
known history of cardiovascular disease, on-going infec-
tion, or any other chronic medical diagnosis.
Cardiovascular health was assessed on the basis of med-
ical history, and those with a no history of cardiovascular
disease were considered healthy in this regard.
Clinical examination for periodontal disease
Patients underwent a comprehensive periodontal exami-
nation including radiographs, and the oral health status
was verified by a thorough clinical examination. A total
of four dentists, all with extensive clinical experience in
the field of periodontology undertook all clinical exami-
nations. Teeth, gums and the oral mucosa were evalu-
ated, and the pocket depth was measured using a
calibrated (in mm) periodontal probe. Probing depth is
the distance between the gingival margin and the bottom
of the gingival pocket measured from six angles of each
tooth. The presence or absence of dental plaque at the
gingival margin along the mesial, buccal, distal and lin-
gual aspects was determined.26 The dental plaque was
made visible by gently moving the tip of the probe along
the gingival margin of the four sides of each tooth.
Gingival pockets 4 mm or deeper were considered to be
pathogenic. Gingival inflammation was noted as bleed-
ing on probing and data are expressed as the proportion
of bleeding sites from the total number of sites in the
dentition.
Clinical treatment of periodontal disease
Patients underwent a thorough periodontal treatment,
including extraction of teeth due to periodontitis, caries
lesions, endodontic or other reasons. Thereafter, oral
hygiene instructions were given, and scaling and root
planing was done. This treatment continued until the
patient obtained good oral hygiene by him/herself and
was free from calculus. After 3 months, the patient was
re-examined according to the baseline examination crite-
ria. Those, who still had pockets deeper than 6 mm at the
re-evaluation, had standardized periodontal flap surgery.
According to normal periodontal treatment, a second
healing process then took place for an additional 3
months. The patient was again re-examined and evalu-
ated. The oral health status including radiographs was veri-
fied by a thorough clinical examination. The investigator
 LPS associates with pro-atherogenic lipoproteins in periodontitis patients 249
who did the base-line examination also did the re-exam-
inations and re-evaluations.
This study was approved by the Regional Ethics
Committee of the Karolinska Institutet and was con-
ducted in accordance with the Helsinki Declaration. All
participants gave their informed consent. Analyses of all
blood samples were performed blindly.
Plasma determinations
Fasting blood was collected into EDTA Vacutainer
tubes. Plasma was obtained after centrifugation at
1500 g for 10 min and stored at –70°C until analysis.
Samples were taken at base-line and after periodontal
treatment on average 6 months after entering the study.
Plasma total cholesterol, triglyceride, LDL, and HDL
cholesterol concentrations as well as apoA-I were deter-
mined using standard clinical chemistry procedures with
a Hitachi 917 analyzer (Roche AG Diagnostics,
Mannheim, Germany). Serum C-reactive protein (CRP)
levels were determined using a high-sensitivity commer-
cial assay kit (DADE Behring, Deerfield, IL, USA).
Serum interleukin (IL)-1β, IL-6, and tumor necrosis fac-
tor (TNF)-α (R&D Systems Europe Ltd, Abingdon, UK)
concentrations were determined according to the manu-
facturers’ recommended protocols. LPS activity of
plasma samples and lipoprotein fractions was deter-
mined by Limulus amebocyte lysate assay coupled with
a chromogenic substrate (HyCult Biotechnology b.v.,
Uden, The Netherlands) on diluted (1:5, v/v in endo-
toxin-free water) samples.
Isolation of lipoproteins
Very low-density plus intermediate-density lipoproteins
(IDL), LDL, HDL2, HDL3, and lipoprotein-deficient
plasma (LPDP) were isolated by sequential ultracen-
trifugation27 using a Beckman Airfuge at 160,000 g at
+4°C. The run time was 2 h for VLDL-IDL, LDL, and
HDL2, and 2.5 h for HDL3. KBr was used to adjust the
Table 1. Clinical parameters before and after periodontal treatment
Clinical parameter Before treatment
Number of teeth 23.2 (5.2)
Visible plaque (%) 53.4 (23.7)
Bleeding on probing (%) 41.1 (14.7)
Number of periodontal pockets > 4 mm 46.3 (17.1)
Depth of deepened (> 4 mm> periodontal pockets 5.74 (0.54)
Teeth with furcation lesions 3.12 (1.98)
Number of mobile teeth 1.12 (1.73)
Values are mean (SD). *Wilcoxon signed ranks test.
density of the samples: VLDL-IDL 1.006–1.019 g/ml,
LDL 1.019–1.063 g/ml, HDL2 1.063–1.12 g/ml, HDL3
1.12–1.21 g/ml and LPDP > 1.21 g/ml. HDL3 and LPDP
fractions were extensively dialyzed against TBS (10 mM
Tris-HCl, pH 7.3, containing 150 mM NaCl) before
determinations. Cholesterol, triglyceride and phospho-
lipid concentrations on lipoprotein fractions were deter-
mined by routine enzymatic methods and LPS activity as
described above.
Statistical analysis
Wilcoxon signed ranks test and Mann–Whitney U-test
were used to test the significance of the differences
between pre- and post-treatment samples, and between
the genders or smokers and non-smokers, respectively.
Interaction between parameters determined was done
using Spearman’s rank correlation. All analyses were
performed with SPSS v.12.0.
RESULTS
The clinical periodontal parameters of the patients
before and after periodontal treatment are summarized in
Table 1. According to all parameters registered, peri-
odontal treatment was successful. The plasma parame-
ters before and after periodontal treatment are presented
in Table 2. Concerning lipoprotein metabolism, there
was a significant increase in HDL cholesterol, apoA-I,
and triglyceride concentration, but no significant change
in total or LDL cholesterol concentration. In addition, no
significant changes were observed in the inflammatory
markers, CRP, IL-1β, TNF-α, or IL-6, after periodontal
treatment.
There was a modest, but statistically significant, increase
in plasma LPS activity after the treatment (Table 2). Plasma
LPS activities did not differ significantly between smokers
and non-smokers, 39.0 ± 16.8 versus 46.4 ± 16.9 EU/ml, or
males and females, 45.6 ± 15.3 versus 42.0 ± 19.3 EU/ml,
respectively. The 22 subjects, whose plasma LPS levels
After treatment P-value*
21.4 (6.3) < 0.001
12.6 (15.6) < 0.001
15.9 (13.5) < 0.001
18.9 (12.5) < 0.001
5.42 (0.69) 0.014
2.58 (2.03) 0.028
0.24 (0.66) 0.004
250 Kallio, Buhlin, Jauhiainen et al.
Table 2. Plasma parameters before and after periodontal treatment

Parameter determined Before treatment After treatment P-value*

Total cholesterol (mmol/l) 5.3 (1.0) 5.5 (1.0) 0.064

HDL cholesterol (mmol/l) 1.28 (0.38) 1.33 (0.40) 0.007
Apolipoprotein A-I (g/l) 1.52 (0.32) 1.63 (0.38) 0.016
Triglycerides (mmol/l) 1.57 (1.13) 1.69 (0.78) 0.039
LDL cholesterol (mmol/l) 3.33 (0.94) 3.41 (1.01) 0.586
CRP (mg/l) 2.17 (2.71) 1.76 (1.83) 0.372
IL-1β (pg/ml) 1.26 (1.56) 0.86 (1.00) 0.087
TNF-α (pg/ml) 4.31 (2.39) 4.51 (2.78) 0.491
IL-6 (pg/ml) 2.93 (2.59) 3.18 (1.49) 0.557
LPS (EU/ml) 44.0 (17.0) 55.7 (24.2) 0.006
LPS/cholesterol (EU/µmol) 8.59 (3.62) 10.39 (4.23) 0.009
LPS/HDL cholesterol (EU/µmol) 40.0 (23.2) 48.8 (29.1) 0.018
LPS/apoA-I (EU/mg) 33.5 (15.9) 41.4 (23.2) 0.025

Values are mean (SD). *Wilcoxon signed ranks test.

(Fig. 1A). The mean recovery percentage of LPS activity in

Fig. 1. LPS distribution among lipoprotein classes. Main lipoprotein
fractions were isolated by sequential ultracentrifugation from 34 patients
with periodontitis before and 6 months after periodontal treatment. LPS
activity was determined from these fractions by Limulus amebocyte lysate
assay, and the LPS distribution was calculated among lipoprotein fractions.
(A) The whole population with 34 subjects. (B) Patients, whose plasma
LPS activity decreased after periodontal treatment, n = 22. *P < 0.05,
mean and SD are shown.
decreased after the periodontal treatment (from 50.0 ± 18.8 to
41.4 ± 17.1; P = 0.002) had higher baseline HDL cholesterol
(1.47 ± 0.47 vs 1.24 ± 0.43 mmol/l; P = 0.044), apoA-I (1.68
± 0.27 vs 1.48 ± 0.32 g/l; P = 0.044), and lower CRP (1.16 ±
1.26 vs 2.64 ± 3.11 mg/l; P = 0.028) and IL-6 concentrations
(1.77 ± 1.61 vs 3.44 ± 2.71 pg/ml; P = 0.011) than the 12 sub-
jects whose LPS levels increased, respectively.
The LPS distribution among the main lipoprotein classes
was determined after their isolation by ultracentrifugation
the samples was 86% (range, 59–121%). The activity of
LPS among the lipoproteins followed the descending order:
VLDL-IDL 41.3 ± 12.1%, LPDP 25.0 ± 7.0%, HDL3 13.1
± 5.2%, LDL 11.5 ± 3.7%, and HDL2 9.2 ± 2.8%. No sig-
nificant differences were observed in the LPS distribution
after periodontal treatment. In those 22 patients, whose LPS
activity decreased after the treatment, a minor non-signifi-
cant decrease was seen in all lipoprotein fractions (Fig. 1B).
The mass compositions of the isolated lipoprotein
classes were determined before and after periodontal treat-
ment (Table 3). There were, however, no notable changes
in the mean values between the time points, except a minor
decrease in the VLDL-IDL phospholipid content. The
VLDL-IDL fraction was highly enriched with cholesterol
(40.9 ± 5.8%) at the cost of triglycerides (16.0 ± 5.1%).
The cholesterol content of LDL was relatively low.
To determine how the variables were related, a corre-
lation analyses was performed (Table 4). Plasma LPS
activity correlated positively with some clinical peri-
odontal parameters, CRP, and triglyceride concentration,
and negatively with HDL cholesterol and apoA-I con-
centrations. Similar correlation coefficients were
obtained for VLDL-bound LPS activity, which, how-
ever, did not correlate with HDL. The correlations
between clinical parameters and LDL or HDL bound
LPS activities were inverse.
DISCUSSION
The main finding of the present study was that, in peri-
odontitis, LPS is associated with the pro-atherogenic
VLDL-IDL fraction, which was surprisingly highly
enriched with cholesterol. Although the periodontal
treatment was clinically successful, it did not cause
 LPS associates with pro-atherogenic lipoproteins in periodontitis patients 251

Table 3. Lipoprotein compositions before and after periodontal treatment

Lipoprotein class Mean mass composition (SD)*

Cholesterol (%) Triglycerides (%) Phospholipids (%) Proteins (%)

VLDL-IDL Before 40.9 (5.8) 16.0 (5.1) 29.0 (4.2)# 14.0 (2.0)
After 41.5 (4.2) 16.5 (5.7) 27.7 (3.6) 14.2 (1.5)
LDL Before 36.1 (4.3) 9.1 (3.0) 40.0 (4.7) 14.9 (1.8)
After 35.2 (4.2) 9.1 (2.8) 41.2 (4.3) 14.5 (1.7)
HDL2 Before 21.7 (2.2) 5.2 (2.0) 33.4 (4.8) 39.7 (4.0)
After 21.3 (2.3) 5.4 (2.2) 34.2 (5.1) 39.1 (4.2)
HDL3 Before 17.0 (3.1) 3.5 (1.5) 34.4 (10.2) 45.1 (8.1)
After 17.0 (2.4) 3.3 (1.2) 34.7 (8.7) 45.1 (6.2)

*Mean percentage of components from the total lipoprotein mass.

#
Wilcoxon signed ranks test, P = 0.049 before vs after treatment.

Table 4. Significant correlation coefficients between the variables determined before treatment

Spearman correlation coefficients

Plasma LPS VLDL–LPS LDL–LPS HDL2–LPS HDL3–LPS Free LPS

Visible plaques 0.360

Depth of deepened
periodontal pockets 0.390 0.345 –0.402 –0.375
Mobile teeth 0.399 0.484 –0.398 –0.474
CRP 0.328 0.367
Cholesterol 0.384 –0.505
HDL cholesterol –0.460 0.354
Triglycerides 0.703 0.427 –0.460 –0.403 –0.380
LDL cholesterol 0.346 –0.412
TNF-α 0.436 –0.433
ApoA-I –0.338

Spearman correlation coefficients with P < 0.05; the non-significant correlation coefficients are excluded.

notable changes in either plasma LPS activity or its dis-
tribution among lipoprotein classes. Plasma or VLDL-
IDL bound LPS had a positive correlation with severity
of periodontitis, CRP, and plasma triglyceride concentra-
tion, and a negative correlation with plasma HDL cho-
lesterol concentration.
In contrast to our earlier study,5 the mean LPS activity
increased in the present group of periodontitis patients
after periodontal treatment. Although the increment was
not striking, the difference reached statistical signifi-
cance. The increase in the mean LPS activity was due to
12 patients, since in 22 patients the LPS activity actually
decreased significantly. Those 22 patients may have had
a better ‘systemic inflammation status’ than those whose
LPS activity increased. This was reflected by higher
HDL cholesterol and lower CRP and IL-6 concentra-
tions at base-line. Clear conclusions cannot, however, be
drawn, since studying the inflammation parameters was
not the main aim of the study and the number of patients
would be too small for analysis.
Compared to published plasma LPS values, the pre-
sent LPS activities were rather low (44 EU/ml), situated
between those found in the healthy elderly with a mean
of 6.7 EU/ml,28 a random sample of the middle-aged
with a mean of 25.4 EU/ml,24 and middle-aged subjects
including cardiovascular disease patients having a
median of 122.8 EU/ml.29 It is, however, difficult to
compare results between different laboratories and pop-
ulations due to the variety of assay kits available and to
the low inter-assay reproducibility of the assays.30 High-
density lipoprotein cholesterol concentration increased
after treatment in the present population, but was not
sufficient to compensate for the increase seen in LPS
activity: also, the LPS/HDL ratio increased significantly
after periodontal treatment. As with LPS levels in the
total population, the inflammation-associated markers
such as CRP, IL-1β, TNF-α, and IL-6, did not decrease
after periodontal treatment. The results suggest that,
despite the successful clinical healing of the patients,
their systemic inflammation status did not respond to the
252 Kallio, Buhlin, Jauhiainen et al.
mechanical treatment applied, thereby reflecting the per-
sistent nature of the disease. Unfortunately, we did not
have bacterial samples from the patients, since base-line
and follow-up microbiology might have shed light on
the healing process.
We found that 25% of the LPS activity existed free in
plasma, which is in accordance with earlier results that
around 80% of plasma total LPS is carried by the
lipoprotein particles.7 On average, 41% of the plasma
LPS activity was bound to the VLDL-IDL fraction and
other lipoprotein classes bound only 34% of the activity.
There were no significant differences in LPS activities
between LDL, HDL2 and HDL3 fractions. The distribu-
tion was, therefore, very similar to that found in patients
with sepsis, although their LPS is more evenly distrib-
uted among VLDL and HDL subpopulations despite the
sharp decrease in HDL cholesterol concentrations in
acute phase response (APR).9,31 The VLDL–LPS com-
plexes are readily taken up by the liver thereby protect-
ing sepsis patients from the deleterious effects of LPS.10
Very low-density lipoprotein strongly supports LPS-
induced production of cytokines,32 which in turn sup-
press the RNA expression level of VLDL receptors.33
Although the following short-term hypertriglyceridemia
is considered beneficial for the sepsis patients, it is an
independent risk factor for atherosclerosis.34,35
The VLDL-IDL fractions characterized before and
after periodontal treatment were highly enriched with
cholesterol (41%), phospholipids (29%), and proteins
(14%), and depleted in triglycerides (16%). The pattern
suggests that a major proportion of the VLDL-IDL frac-
tion was composed of IDL-class particles. These compo-
sitional data for VLDL are quite different to those
reported for the ‘normal’ VLDL composition (27%,
12%, 8%, and 50%, respectively).36 The VLDL compo-
sition during a chronic infection, like periodontitis, has
not been studied earlier, and also APR-VLDL has gained
only scarce attention in the literature. Kitchens and
Thompson,31 however, have presented preliminary data
that these lipoproteins may contain higher than normal
proportions of cholesterol. Their data suggest that these
lipoproteins can replace HDL as the dominant LPS
acceptor during sepsis, when HDL levels are low.31 In
the present study, the HDL cholesterol levels were also
quite low: 80% of the subjects had HDL cholesterol
lower than 1.5 mmol/l and 18% lower than 1.0 mmol/l.
In addition, VLDL–LPS had a strong negative correla-
tion with HDL2–LPS and HDL3–LPS (r = –0.601 and
–0.754, respectively; P < 0.001).
Our study raises several interesting questions. The
most important is the fate of LPS–VLDL. According to
some in vitro studies, by binding to VLDL, LPS promotes
the uptake of the particles by macrophages37 thereby
increasing their pro-atherogenic potency. The particle
size, however, has been observed to be crucial for the
uptake in vivo. In hypercholesterolemic rabbits, the cut-
off limit for arterial wall uptake of lipoproteins was 75
nm.38 Since the VLDL particle size generally ranges
approximately between 30–80 nm, the largest particles
may escape entering the intima. The limitation of our
study is that we did not have an opportunity to determine
the VLDL-IDL particle sizes. The composition, how-
ever, suggests that they represent a small-sized VLDL
subpopulation. Another question is whether LPS
remains associated with apoB-particles during their
lipolytic processing up to the LDL stage. To our knowl-
edge, there is no information on the lipase activity in
post-heparin plasma of periodontitis patients. During
APR, however, both lipoprotein and hepatic lipase activ-
ity decreases.39
A high serum LPS activity is found to be an indepen-
dent risk factor for atherosclerosis.24,40 In our earlier
study, a high serum LPS activity predicted incident car-
diovascular disease events, when it was associated with
a low HDL cholesterol concentration or high CRP or IL-
6.29 Periodontitis patients have been observed to suffer
from endotoxemia,3–5 but without specific techniques to
determine LPS concentrations derived from periodontal
pathogens, definite conclusions cannot be drawn.
Induction of systemic inflammation by LPS has been
proposed to be the pathogenic mechanism behind the
association of infections and atherosclerosis. Therefore,
as suggested by our results, LPS may play a part in peri-
odontitis-induced atherosclerosis as well.
ACKNOWLEDGEMENT
The work was financially supported by the Academy of
Finland (grant 118391 to PJP).
REFERENCES
1. Knuuttila M. Hampaiden kiinnityskudossairaudet. In: Suominen-
Taipale L, Nordbland A, Vehkalahti M, Aromaa A. (eds)
Suomalaisten aikuisten suunterveys, Terveys 2000–tutkimus.
Finland: National Public Health Institute, 2004; B16; 88–97.
2. Kononen E, Paju S, Pussinen PJ et al. Population-based study of
salivary carriage of periodontal pathogens in adults. J Clin
Microbiol 2007; 45: 2446–2451.
3. Silver JG, Martin AW, McBride BC. Experimental transient
bacteraemias in human subjects with varying degrees of plaque
accumulation and gingival inflammation. J Clin Periodontol
1977; 4: 92–99.
4. Geerts SO, Nys M, De MP et al. Systemic release of endotoxins
induced by gentle mastication: association with periodontitis
severity. J Periodontol 2002; 73: 73–78.
5. Pussinen PJ, Vilkuna-Rautiainen T, Alfthan G et al. Severe
periodontitis enhances macrophage activation via increased
serum lipopolysaccharide. Arterioscler Thromb Vasc Biol 2004;
24: 2174–2180.
6. Levels JH, Abraham PR, van den Ende A, van Deventer SJ.
 LPS associates with pro-atherogenic lipoproteins in periodontitis patients 253
Distribution and kinetics of lipoprotein-bound endotoxin. Infect
Immun 2001; 69: 2821–2828.
7. Harris HW, Johnson JA, Wigmore SJ. Endogenous lipoproteins
impact the response to endotoxin in humans. Crit Care Med
2002; 30: 23–31.
8. Levine DM, Parker TS, Donnelly TM, Walsh A, Rubin AL. In
vivo protection against endotoxin by plasma high density
lipoprotein. Proc Natl Acad Sci USA 1993; 90: 12040–12044.
9. Levels JH, Lemaire LC, van den Ende AE, van Deventer SJ, van
Lanschot JJ. Lipid composition and lipopolysaccharide binding
capacity of lipoproteins in plasma and lymph of patients with
systemic inflammatory response syndrome and multiple organ
failure. Crit Care Med 2003; 31: 1647–1653.
10. Barcia AM, Harris HW. Triglyceride-rich lipoproteins as agents of
innate immunity. Clin Infect Dis 2005; 41 (Suppl 7): S498–S503.
11. Brown MS, Goldstein JL. Lipoprotein metabolism in the macro-
phage: implications for cholesterol deposition in atherosclerosis.
Annu Rev Biochem 1983; 52: 223–261.
12. Lakio L, Lehto M, Tuomainen AM et al. Pro-atherogenic
properties of lipopolysaccharide from the periodontal pathogen
Actinobacillus actinomycetemcomitans. J Endotoxin Res 2006;
12: 57–64.
13. Stoll LL, Denning GM, Weintraub NL. Potential role of
endotoxin as a proinflammatory mediator of atherosclerosis.
Arterioscler Thromb Vasc Biol 2004; 24: 2227–2236.
14. Mattila KJ, Nieminen MS, Valtonen VV et al. Association
between dental health and acute myocardial infarction. BMJ
1989; 298: 779–781.
15. Beck J, Garcia R, Heiss G, Vokonas PS, Offenbacher S.
Periodontal disease and cardiovascular disease. J Periodontol
1996; 67: 1123–1137.
16. Howell TH, Ridker PM, Ajani UA, Hennekens CH, Christen
WG. Periodontal disease and risk of subsequent cardiovascular
disease in U.S. male physicians. J Am Coll Cardiol 2001; 37:
445–450.
17. Desvarieux M, Demmer RT, Rundek T et al. Periodontal
microbiota and carotid intima-media thickness: The oral
infections and vascular disease epidemiology study (INVEST).
Circulation 2005; 111: 576–582.
18. Spahr A, Klein E, Khuseyinova N et al. Periodontal infections
and coronary heart disease: role of periodontal bacteria and
importance of total pathogen burden in the coronary event and
periodontal disease (CORODONT) study. Arch Intern Med 2006;
166: 554–559.
19. Pussinen PJ, Nyyssonen K, Alfthan G, Salonen R, Laukkanen
JA, Salonen JT. Serum antibody levels to Actinobacillus
actinomycetemcomitans predict the risk for coronary heart
disease. Arterioscler Thromb Vasc Biol 2005; 25: 833–838.
20. Li L, Messas E, Batista EL, Levine RA, Amar S. Porphyromonas
gingivalis infection accelerates the progression of atherosclerosis
in a heterozygous apolipoprotein E-deficient murine model.
Circulation 2002; 105: 861–867.
21. Lalla E, Lamster IB, Hofmann MA et al. Oral infection with a
periodontal pathogen accelerates early atherosclerosis in
apolipoprotein E-null mice. Arterioscler Thromb Vasc Biol 2003;
23: 1405–1411.
22. Brodala N, Merricks EP, Bellinger DA et al. Porphyromonas
gingivalis bacteremia induces coronary and aortic atherosclerosis
in normocholesterolemic and hypercholesterolemic pigs.
Arterioscler Thromb Vasc Biol 2005; 25: 1446–1451.
23. Tuomainen AM, Jauhiainen M, Kovanen PT, Metso J, Paju S,
Pussinen PJ. Aggregatibacter actinomycetemcomitans induces
MMP-9 expression and proatherogenic lipoprotein profile in
apoE-deficient mice. Microb Pathog 2008; 44: 111–117.
24. Wiedermann CJ, Kiechl S, Dunzendorfer S et al. Association of
endotoxemia with carotid atherosclerosis and cardiovascular
disease: Prospective results from the Bruneck study. J Am Coll
Cardiol 1999; 34: 1975–1981.
25. Nyman S, Lindhe J. Clinical periodontology and implant
dentistry. In: Lindhe J, Karring T, Lang NP. (eds) Clinical
Periodontology and Implant Dentistry, 4th edn. Copenhagen:
Blackwell Munksgaard, 2003; 403–413.
26. Love WD, Ramirez JM, Fultz RP. An oral hygiene measurement
system for possible research and clinical use. J Public Health
Dent 1975; 35: 227–230.
27. Havel RJ, Eder HA, Bragdon JH. The distribution and chemical
composition of ultracentrifugally separated lipoproteins in human
serum. J Clin Invest 1955; 34: 1345–1353.
28. Goto T, Eden S, Nordenstam G, Sundh V, Svanborg-Eden C,
Mattsby-Baltzer I. Endotoxin levels in sera of elderly individuals.
Clin Diagn Lab Immunol 1994; 1: 684–688.
29. Pussinen PJ, Tuomisto K, Jousilahti P, Havulinna AS, Sundvall J,
Salomaa V. Endotoxemia, immune response to periodontal
pathogens, and systemic inflammation associate with incident
cardiovascular disease events. Arterioscler Thromb Vasc Biol
2007; 27: 1433–1439.
30. Guidet B, Barakett V, Vassal T, Petit JC, Offenstadt G.
Endotoxemia and bacteremia in patients with sepsis syndrome in
the intensive care unit. Chest 1994; 106: 1194–1201.
31. Kitchens RL, Thompson PA, Munford RS, O’Keefe GE. Acute
inflammation and infection maintain circulating phospholipid
levels and enhance lipopolysaccharide binding to plasma
lipoproteins. J Lipid Res 2003; 44: 2339–2348.
32. Stollenwerk MM, Schiopu A, Fredrikson GN, Dichtl W, Nilsson
J, Ares MP. Very low density lipoprotein potentiates tumor
necrosis factor-alpha expression in macrophages. Atherosclerosis
2005; 179: 247–254.
33. Jia L, Takahashi M, Morimoto H et al. Changes in cardiac lipid
metabolism during sepsis: the essential role of very low-density
lipoprotein receptors. Cardiovasc Res 2006; 69: 545–555.
34. Bansal S, Buring JE, Rifai N, Mora S, Sacks FM, Ridker PM.
Fasting compared with nonfasting triglycerides and risk of
cardiovascular events in women. JAMA 2007; 298: 309–316.
35. Nordestgaard BG, Benn M, Schnohr P, Tybjaerg-Hansen A.
Nonfasting triglycerides and risk of myocardial infarction,
ischemic heart disease, and death in men and women. JAMA
2007; 298: 299–308.
36. Skipski VP, Barclay M, Barclay RK, Fetzer VA, Good JJ,
Archibald FM. Lipid composition of human serum lipoproteins.
Biochem J 1967; 104: 340–352.
37. Funk JL, Feingold KR, Moser AH, Grunfeld C.
Lipopolysaccharide stimulation of RAW 264.7 macrophages
induces lipid accumulation and foam cell formation.
Atherosclerosis 1993; 98: 67–82.
38. Nordestgaard BG, Zilversmit DB. Hyperglycemia in
normotriglyceridemic, hypercholesterolemic insulin-treated
diabetic rabbits does not accelerate atherogenesis.
Atherosclerosis 1988; 72: 37–47.
39. Sammalkorpi K, Valtonen V, Kerttula Y, Nikkila E, Taskinen
MR. Changes in serum lipoprotein pattern induced by acute
infections. Metabolism 1988; 37: 859–865.
40. Kiechl S, Egger G, Mayr M et al. Chronic infections and the risk
of carotid atherosclerosis: prospective results from a large
population study. Circulation 2001; 103: 1064–1070.