Abstract

A

J

, G

B

, E

G

Seropositivity for

Chlamydophila pneumoniae

is Associated with Increased Body Mass Index

and Serum Lipid Levels: Evidence from the Sample

Urban Population in Bydgoszcz*

Wpływ zakażeń

Chlamydophila pneumoniae

na wskaźniki otyłości

i gospodarki lipidowej u osób dorosłych z terenu Bydgoszczy

1_{Department of Bromatology and Molecular Nutrition, Faculty of Pharmacy, Collegium Medicum,}

Nicolaus Copernicus University, Bydgoszcz, Poland

2_{Department of Microbiology, Faculty of Pharmacy, Collegium Medicum, Nicolaus Copernicus University,}

Bydgoszcz, Poland

Adv Clin Exp Med 2007, 16, 6, 743–749 ISSN 1230−025X

ORIGINAL PAPERS

© Copyright by Silesian Piasts University of Medicine in Wrocław

Abstract

Background.Although it is well known that obesity is a multifactoral disease, the possibility that some viruses or bacteria might be a serious etiologic factor in this metabolic disorder has received relatively little attention.

Objectives.As no information is available on a link between Chlamydophila pneumoniaeinfections and the pre− valence of obesity in Poland, this study was conducted to provide such pilot data from a sample of professionally active adults randomly recruited from an urban area in Bydgoszcz.

Material and Methods. Anthropometric measurements were obtained from 39 healthy middle−aged adult subjects (4 male, 35 female) of whom 27 were obese or overweight. Antibody status (IgG, IgA, IgM) was determined by enzyme−linked immunosorbent assay (ELISA). The total cholesterol (TChol) and triglyceride (TG) concentrations in serum were performed by standard enzymatic methods.

Results. The group of subjects with IgG antibodies for C. pneumoniae was characterized by higher BMI (31.4 ± 5.5

vs. 27.4 ± 6.3 kg/m2_{, p= 0.038), higher serum TChol (5.64 ± 0.91vs. 5.02 ± 0.81 mmol/l, p= 0.047), and higher}

TG level (1.38 ± 0.34vs. 1.16 ± 0.49 mmol/l, p= 0.047) than seronegative subjects. Seropositivity for C. pneumo− niae IgA antibodies was associated with higher serum TChol concentration (5.91 ± 0.67vs. 5.15 ± 0.93 mmol/l,

p= 0.007). No subjects were positive for IgM antibodies.

Conclusions. The results of this pilot study indicate thatC. pneumoniae infection is associated with higher body mass index and elevated serum lipid concentrations in a sample of a middle−aged adult urban population from Byd− goszcz (Adv Clin Exp Med 2007, 16, 6, 743–749).

Key words:Chlamydophila pneumoniae, infectobesity, BMI, serum cholesterol, serum triglycerides.

Streszczenie

Wprowadzenie. Otyłość jest jednostką chorobową o złożonym i wieloczynnikowym podłożu, możliwość jednak udziału czynnika infekcyjnego w etiologii otyłości była rzadko rozważana, a w Polsce nie podejmowano dotych− czas tego problemu.

Cel pracy. Określenie współzależności między obecnością w surowicy przeciwciał swoistych dla Chlamydophila pneumoniae a występowaniem otyłości i gospodarką lipidową aktywnych zawodowo osób dorosłych stanowiących próbkę populacji wielkomiejskiej z terenu Bydgoszczy.

Materiał i metody.W grupie 39 dorosłych osób w średnim wieku (w tym 27 z nadwagą lub otyłością) objętych badaniami przeprowadzono pomiary antropometryczne. Stężenie cholesterolu całkowitego (TChol) i triglicerydów (TG) w surowicy badanych osób oceniono z zastosowaniem metody enzymatycznej. Do oznaczenia obecności przeciwciał klasy IgG, IgA, IgM dla C. pneumoniae wykorzystano metodę immunoenzymatyczną.

According to reports from different areas, the prevalence of overweight and obesity is increasing at an alarming rate both in developed and develo− ping countries. However, the estimated prevalence rates of obesity vary worldwide [1–4]. The most recent WHO data show that about 20% men and 30% women in European countries are obese [5]. According to a recent national survey in Poland, over 50% of adults (19–59 years old) are overwe− ight or obese [6]. The classical treatment of obesi− ty based on increased physical activity and decre− ased calorie intake has not been successful. The lack of efficacy of this therapeutic approach is pro− bably due to an incomplete understanding of the precise etiology of obesity [7, 8]. The etiology of obesity is multifactorial, as interactions between nutritional, metabolic, cultural, behavioral, social, and genetic factors are involved in the develop− ment of obesity [1, 9–11]. There are also a number of different lines of evidence indicating that vario− us infections may be linked to obesity [12–16]. However, the possibility that viruses or bacteria might be etiologic agents in human obesity has re− ceived relatively little attention. One of these agents is Chlamydophila pneumoniae, an intracel− lular Gram−negative bacterium that commonly causes acute as well as chronic respiratory infec− tion [17]. It was suggested that C. pneumoniaein− fection may be associated with an increase in bo− dy mass index [15, 16]. There are also accumula− ting data suggesting that C. pneumoniaeinfection affects serum lipid concentrations [18–20].

The present study investigated the relationship between overweight and lipid profile and evidence of C. pneumoniae antibodies in a sample middle− aged urban population from Bydgoszcz (Poland). A relationship between C. pneumoniae infection and asthma [21] and abdominal aortic aneurysm [22] was observed in some Polish populations; ho− wever, the present authors found no information available on such an association with indices of obesity in any social group in Poland and this stu− dy was conducted to provide such pilot data.

Methods

Participants

The studied population consisted of 39 adult subjects of whom four (10%) were male. The stu− dy and control groups were recruited from the lo− cal community (Bydgoszcz, Poland) by advertise− ments in local pharmacies during the period from March 2006 to April 2007. The subjects were in− formed about the aim of this study and signed a written consent form approved by local bioethics committee. The participants were classified accor− ding to their BMI (kg/m2_{) status as normal weight}

(group A: control subjects) and overweight or obe− se (group B: studied subjects). As recommended by WHO, normal weight was defined as a BMI < < 25 kg/m2_{, overweight as a BMI} ≥_{25 kg/m}2_{, and}

obesity as a BMI ≥30 kg/m2_{[1]. Anthropometric}

measurements, which included height, weight, and four skinfold thickness measurements (triceps, bi− ceps, subscapular, and suprailiac), were used to calculate the body mass index (BMI, kg/m2_{) and}

the percentage of fat mass (%FM, %) [23]. Total body fat was calculated using the Durnin and Wo− mersley formula [24]. These anthropometric data were obtained by, respectively, a height scale (Ra− dwag, Poland), a digital electronic weighing scale (Radwag), and an electronic skinfold caliper (Skyndex I, USA).

Biochemical Analyses

Venous blood was drawn after overnight fa− sting and the serum was separated from the red cells by centrifugation at 3000 ×gfor 10 minutes. The serum was immediately divided into aliquots and frozen at –40°C until the serological assays were performed. Total serum cholesterol (TChol, mmol/l) and triglyceride (TG, mmol/l) levels were determined by standard enzymatic methods with reagents from Limarco, Poland. The analyses we-re performed on an automated BTS 310 analyzer (Biosystems, Barcelona, Spain) in an accredited analytical laboratory.

Wyniki.Odnotowano, że osoby ze stwierdzoną obecnością przeciwciał klasy IgG charakteryzowały się statystycz− nie istotnie wyższym BMI (31,4 ± 5,5vs. 27,4 ± 6,3 kg/m2_{; p= 0,038) oraz stężeniem TChol (5,64 ± 0,91vs. 5,02 ±}

± 0,81 mmol/l; p= 0,047) i TG (1,38 ± 0,34vs. 1,16 ± 0,49 mmol/l; p= 0,047) w surowicy niż osoby seronega− tywne. Wykazano również, że obecność w surowicy przeciwciał klasy IgA jest związana z istotnie większym stę− żeniem TChol (5,91 ± 0,67vs. 5,15 ± 0,93 mmol/l; p= 0.007). Nie stwierdzono obecności przeciwciał klasy IgM w badanej grupie osób z terenu Bydgoszczy.

Wnioski. Uzyskane wstępne wyniki wskazują na duże prawdopodobieństwo udziału czynnika infekcyjnego w etiologii nadwagi i otyłości. Wykazano również współzależność między obecnością przeciwciał swoistych dla

C. pneumoniae a profilem lipidowym w surowicy badanych osób z terenu Bydgoszczy (Adv Clin Exp Med 2007, 16, 6, 743–749).

Serological Analyses

The analyses of the IgG, IgA, and IgM Chla− mydophila pneumoniae antibodies were perfor− med using a commercially available enzyme−lin− ked immunosorbent assay (ELISA, Vircell, Spain) according to Gutierrez et al. [25]. First, 5 µl of pa− tient serum, negative, positive, and cut−off con− trols, and 100 µl of serum diluent were added to each well (phosphate buffer). The microplate was covered and incubated for 45 min at 37C. After incubation the strips was washed five times with 300 µl of washing solution (phosphate buffer). To each well, 100 µl of conjugate (peroxidase labeled anti-human IgG, IgM, or IgA antibodies) was ad-ded and the plate was incubated for 30 min at 37°C. The plate was washed as described above. To each well, 100 µl of substrate solution (TMB, tetramethylbenzidine) was added and the plate was incubated at room temperature for 25 min. Finally, 50 µl of stop reagent (0.5 M H2SO4) was added to

each well and the optical density was measured. Absorbance was read at 450 nm by an ELISA Mi-crowell Plate Reader (Synergy HT, Bio-Tek In-struments Inc., USA). The results were determined by calculating an index value from the optical den-sity values relative to control materials [25]. The index value was calculated by dividing the pa-tient’s absorbance value by the mean absorbance value of the cut-off point and then multiplied by 10. As recommended by the manufacturer, an

ELISA test index value > 11.0 was considered po-sitive and < 9.0 negative [25].

Statistical Calculations

The results are given as the mean and standard deviation or as a number (%). Differences between the groups were tested by χ2_{for categorical variables}

and by the Mann Whitney U−test and Pearson’s cor− relation for continuous variables. Apvalue ≤0.05 was considered statistically significant. The statisti− cal analyses were carried out by STATISTICA PL v. 6.1 software (Stat−Soft, Inc., Tulsa, OK, USA).

Results

The anthropometrical and biochemical charac− teristics of the subjects and their serological status are shown in Table 1. Of the 39 participants, 12 (30.8%) were of normal weight (group A) and 27 (69.2%) were overweight or obese (group B) (Table 1). All the male subjects were classified in− to group B. It was observed that the overwei− ght/obese participants in group B had a higher fre− quency of IgG and IgA seropositivity than the non− obese subjects in group A. The prevalence of serum IgG antibody for C. pneumoniaewas 41.7% in group A and 70.4% in group B and the presen− ce of C. pneumoniaeIgA antibodies was observed in 16.7% of the non−obese group and in 40.7% of

Parameters Non−obese, group A Overweight or obese, group B p

(Wskaźniki) (Z wagą należną, grupa A) (Otyli lub z nadwagą, grupa B)

n= 12 (30.8%) n= 27 (69.2%)

Age – years) 37.7 ± 16.2 46.8 ± 11.9 0.125

(Wiek – lata)

BMI – kg/m2 _{22.7 ± 1.6 33.1 ± 4.5 0.000}

%FM – % 31.9 ± 5.9 41.3 ± 5.3 0.000

TChol – mmol/l 5.19 ± 1.0 5.49 ± 0.87 0.298

TG – mmol/l 1.02 ± 0.3 1.41 ± 0.4 0.008

IgG positive – % 41.7 70.4 0.089

(IgG dodatni – %)

IgA positive (%) 16.7 40.7 0.140

(IgA dodatni – %)

IgG and IgA positive (%) 16.7 37.0 0.203

(IgG i IgA dodatni – %)

Table 1. Characteristics of the studied populations

Tabela 1. Charakterystyka grupy badanej

± SD– standard deviation.

p– statistical difference between groups A and B.

n– number of subjects (%). ± SD – odchylenie standardowe.

p– współczynnik istotności różnicy między grupami A i B.

the overweight/obese. No subjects were positive for IgM. Combined seropositivity for both IgG and IgA antibodies suggestive of chronic infection were present in 16.7% of the control group and 83.3% of the study group. Comparison of the non− obese group and the overweight/obese group sho− wed a significantly higher BMI, percentage of to− tal body fat, waist/hip ratio, and serum triglyceride level in the overweight/obese subjects. There were no differences between the groups regarding age and serum cholesterol (Table 1).

A significant relationship was observed be− tween seropositivity for C. pneumoniae and selec−

ted variables in all subjects (Tables 2 and 3). In those subjects who were seropositive forC. pneu− moniae IgG antibodies, the mean BMI was signi− ficantly higher than in the seronegative group (31.4 ± 5.5vs. 27.4 ± 6.3 kg/m2_{, p= 0.038). A si−}

milar association was also observed for C. pneu− moniaeIgA antibody status, but this tendency was not statistically significant. Subjects seropositive forC. pneumoniae IgG and IgA antibodies had si− gnificantly higher serum total cholesterol concen− trations than seronegative subjects (5.64 ± 0.91vs.

5.02 ± 0.81 mmol/l, p= 0.047, and 5.91 ± 0.67vs.

5.15 ± 0.93 mmol/l, p= 0.007, respectively). Sero−

Parameters IgG Positive IgG Negative p

(Wskaźniki) (IgG+_{seropozytywni) (IgG}−_{seronegatywni)}

n = 15 (38.46%) n= 24 (61.53%)

Age – years 48.0 ± 12.6 39.5 ± 15.4 0.097

(Wiek – lata)

BMI – kg/m2 _{31.4 ± 5.5 27.4 ± 6.3 0.038}

%FM – % 40.5 ± 5.2 35.2 ± 8.2 0.076

TChol – mmol/l 5.64 ± 0.91 5.02 ± 0.81 0.047

TG – mmol/l 1.38 ± 0.34 1.16 ± 0.49 0.047

Table 2. Characteristics of the participants with positive and negative serology for

C. pneumoniaeIgG antibody

Tabela 2. Wskaźniki antropometryczne i lipidowe a częstość wykrywania swoistych prze− ciwciał anty−C. pneumoniaeklasy IgG

± SD– standard deviation.

p– statistical difference between groups.

n– number of subjects (%). ± SD – odchylenie standardowe.

p– współczynnik istotności różnicy między grupami IgA−_{i IgA}+_.

n– liczność grupy (%).

Parameters IgG Positive IgG Negative p

(Wskaźniki) (IgG+_{seropozytywni) (IgG}−_{seronegatywni)}

n = 13 (3.3%) n= 26 (66.6%)

Age – years 52.2 ± 9.9 40.0 ± 13.9 0.008

(Wiek – lata)

BMI – kg/m2 _{31.6 ± 5.9 29.0 ± 6.2 0.208}

%FM – % 40.7 ± 5.3 37.3 ± 7.5 0.168

TChol – mmol/l 5.91 ± 0.67 5.15 ± 0.93 0.007

TG – mmol/l 1.37 ± 0.32 1.26 ± 0.45 0.267

Table 3.Characteristics of the participants with positive and negative serology for

C. pneumoniae IgA antibody

Tabela 3. Wskaźniki antropometryczne i lipidowe a częstość wykrywania swoistych prze− ciwciał anty−C. pneumoniaeklasy IgA

± SD– standard deviation.

p – statistical difference between groups.

n– number of subjects (%). ± SD – odchylenie standardowe.

p– współczynnik istotności różnicy między grupami IgA–_{i IgA}+_.

positivity for C. pneumoniae IgG antibodies was also associated with serum triglyceride concentra− tion (TG), i.e. subjects with C. pneumoniae IgG antibodies had significantly higher TG levels (1.38 ± 0.34vs. 1.16 ± 0.49 mmol/l, p= 0.047). There were no differences in mean age between C. pneu− moniaeIgG antibody seropositive and seronegati− ve subjects. However, seropositivity for C. pneu− moniae IgA antibody was associated with age; in this case, seropositive subjects were older than se− ronegative (52.2 ± 9.9vs. 40.0 ± 13.9, p= 0.008) (Tables 2 and 3).

Positive correlations between C. pneumoniae

IgA antibody status and TChol (r= 0.42), and age (r= 0.42) were observed. There were revealed al− so positive associations between IgG antibody sta− tus and TChol (r= 0.32), TG (r= 0.32), and BMI (r= 0.34) (Table 4).

Discussion

The results of this study demonstrated that subjects with positive serology for C. pneumoniae

IgG antibodies are characterized by higher BMI as well as higher serum triglyceride and total chole− sterol concentrations than those of the seronegati− ve group, whereas seropositivity for C. pneumo− niae IgA antibodies was associated with greater age as well as higher total cholesterol concentra− tion. The findings of this study are consistent with the observations of Laurila et al. [18, 26] and Mur− ray et al. [19] of raised serum total cholesterol and triglyceride concentrations in randomly selected samples of Finish and Irish adult male subjects, re− spectively, with serological evidence of C. pneu− moniae infection.

The data of the present study also show that the prevalence of IgG and IgA antibodies and their combined positive serology for C. pneumoniae is about threefold higher in obese or overweight per− sons than in non−obese individuals. There are two possible explanations for the associations between BMI and antibody status observed here. It is possi− ble that infection with C. pneumoniae might be re− lated to increased BMI. A second possibility is that the degree of obesity might be a marker for greater susceptibility to C. pneumoniaeinfection. Howe− ver, it seems impossible that obese people would be exposed to C. pneumoniaemore often than normal weight individuals. It was observed that overwei− ght preadolescents were more susceptible to respi− ratory infections [26]. However, there are also data suggesting that increased BMI is associated with impaired immunity [27–29]. Obesity is a predictor of poorer antibody response to the hepatitis B vac− cine [30, 31]. These findings induced Dhurandhar et al. [12] to suggest that overweight or obese peo− ple would be less likely to produce antibodies when exposed to infection factors than normal−weight people. Thus the prevalence of antibodies should be similar or lower in obese than in lean people. Evidence for an association of C. pneumoniaewith increased BMI in an urban population from Austra− lia was also provided by Dart et al. [16], who obse− rved significant differences in BMI between adult subjects with coronary heart disease classified po− sitive or negative for C. pneumoniae IgG antibo− dies. It should also be noted that in the above study [16], positive serology to other Chlamydia species (C. psittaci andC. trachomatis) was extremely low in the study group. Toplak et al. [15] revealed that in a sample of elderly, non−diabetic subjects recru− ited from Graz (Austria), the prevalence of seroac−

Table 4.Spearman’s Rank correlation coefficients between anthropometric, lipid, and immunological status parameters

Tabela 4.Wartości współczynika Spearmana i współczynnika istotności cha− rakteryzujące obserwowane korelacje między wskaźnikami antropometryczny− mi, lipidowymi i immunologicznymi

Parameters IgG status IgA status

(Wskaźniki) _{Spearman p Spearman p}

r r

Age – year 0.27 0.096 0.42 0.008

(Wiek – lata)

BMI – kg/m2 _{0.34 0.035 0.21 0.204}

%FM – % 0.29 0.072 0.23 0.164

TChol – mmol/l 0.32 0.046 0.42 0.007

TG – mmol/l 0.32 0.046 0.18 0.269

Significant correlations are marked in bold numbers.

tive chlamydial infection was significantly higher in the considerably overweight patients (BMI > 30 kg/m2_{) compared with both moderately obese}

and lean subjects. On the other hand, Ekesbo et al. [32] found that combined seropositivity for

C. pneumoniaeand Helicobacter pyloriwas highly associated with obesity in a group of middle−aged hypertensive patients living in an urban area in Sweden. Quite recently, a similar relationship was shown by Thjodleifsson et al. [33], who reported a significant positive association between being overweight (BMI > 25 kg/m2_{) and IgG antibody}

status for C. pneumoniaeand Helicobacter pylori

in a large multinational urban population study of healthy middle−aged subjects from Iceland, Swe− den, and Estonia. They suggested that infections with C. pneumoniae and Helicobacter pylori are both separately and synergistically associated with overweight and this association is not related to in− dicators of systemic inflammation such as C−reac− tive protein (CRP) or serological markers related with cagA protein and Toxoplasmosa gondii, hepa− titis A virus (HAV), Esptein−Barr virus (EBV), her− pes simplex virus1 (HSV−1), and cytomegalovirus (CMV) infection.

The authors conclude that the observations of this study support the hypothesis that infections, in this case C. pneumoniaeinfection, may be linked to increased BMI. However, as this is hardly an obse−

rvational pilot study, these findings should still be regarded only as suitable for generating working hypotheses, mainly for women. The most serious limitations of this study are the small sample size, the disproportion in the sizes of the study and con− trol groups, the female overrepresentation in both these groups, and the lack of strictly age− and sex− matched controls, so the authors are aware that the presented results may not be amenable for genera− lization. More detailed mechanisms by which

C. pneumoniaeincreases body weight are still unk− nown, and the possibility cannot be excluded that persons with overweight or obesity have an increa− sed susceptibility to C. pneumoniaeinfection. Ho− wever, these findings, despite the above−mentioned shortcomings and limitations, are highly consistent with the main conclusions of other studies perfor− med previously in some European countries [15, 32, 33] and Australia [16] and should encourage the design of a more advanced age− and sex−mat− ched investigation to determine the range of health status, sociodemographic, and socioeconomic fac− tors modifying the possible association between BMI and C. pneumoniaeinfection in urban and ru− ral populations. To the authors’ best knowledge, this pilot study is the first reported evidence of in− fection−related obesity in Poland and the fourth report in Europe, besides Austria [15], Sweden [32, 33], Iceland [33], and Estonia [33].

Acknowledgements. The financial support of this study by the internal research grant CM−UMK−BW−68−2006 is gratefully acknowledged.

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Address for correspondence:

Grzegorz Bazylak

Department of Bromatology and Molecular Nutrition Faculty of Pharmacy

Collegium Medicum

Nicolaus Copernicus University Jagiellońska 13

85−067 Bydgoszcz Poland

Tel.: +48 52 585 39 15 E−mail: [email protected]

Conflict of interest: None declared