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Technical Briefs |
(
1
Department of Internal Medicine, Medical Policlinic,
2
Institute of Clinical Chemistry, and
3
Department of Angiology, University Hospital, CH-8091 Zurich, Switzerland;
aaddress correspondence to this author at: Department of Internal Medicine, University Hospital, CH-8091 Zurich, Switzerland; fax 0041-1-255 45 67, e-mail georg.schulthess{at}dim.usz.ch)
Atherosclerosis, with its different clinical manifestations, is the major cause of morbidity and mortality in the developed countries. Chlamydia pneumoniae infection and the ensuing chronic inflammation have been claimed to contribute to the atherosclerotic process (1). Concurrently, epidemiologic evidence suggests that hyperhomocysteinemia is associated with an increased atherosclerotic risk (2). Both C. pneumoniae infection and hyperhomocysteinemia have been assumed to increase the atherosclerotic risk independently of each other and independently of the classic risk factors.
In vitro, the growth of C. pneumoniae is enhanced in serum-free media and particularly by the depletion of lysine or methionine (3). In human metabolism, homocysteine is produced by demethylation of methionine, and defects in the recycling pathway (in which homocysteine is remethylated to methionine) are one cause of hyperhomocysteinemia (4). This constellation prompted us to investigate whether C. pneumoniae seropositivity and plasma homocysteine concentrations are related in patients with established atherosclerosis.
Approval for this project was obtained from the ethics committee of the University Hospital of Zurich. All patients gave informed consent to participate. The study subjects consulted our ward for follow-up visits and were recruited consecutively. Inclusion criteria were that subjects had to be male and have had an established atherosclerosis defined as the following: (a) history of myocardial infarction or coronary revascularization, (b) history of stroke or carotid artery surgery, or (c) history of angioplasty or bypass surgery of peripheral arteries. Exclusion criteria were diabetes mellitus, malignant neoplasia, current or history of chronic inflammatory disorders, renal insufficiency with serum creatinine >180 µmol/L, and any acute illness.
The sera were tested for antibodies against C. pneumoniae by microimmunofluorescence (MRL) independently of all other laboratory work. The analyses were performed at dilutions of 1:64 (by volume) and higher for IgG and at 1:32 (by volume) and higher for IgA. Fasting serum homocysteine and vitamin B6 were determined with HPLC (Bio-Rad).
Statistical evaluation was performed on an IBM PC using Excel and Statistica (Ver. 4.5) as the software. Multiple regression analysis on serum homocysteine was performed including the C. pneumoniae serostatus and other variables possibly influencing serum homocysteine. The values of C-reactive protein, interleukin 6, and tumor necrosis factor-
were transformed to natural logarithms to approximate a gaussian distribution. Variables with uncertain influence on serum homocysteine (P >0.1) were excluded in the stepwise regression analysis. The comparison of further variables was done using the t-test or the 
2 test.
Of 100 consecutively recruited patients, 51 tested positive for antibodies recognizing C. pneumoniae, and 46 tested negative. The seropositive patients all had IgG titers of at least 1:128. Neither IgG nor IgA positivity was detectable for the seronegative patients. Three patients were excluded from statistical analysis because of undetermined serostatus. No significant differences were found between the two groups regarding age, body mass index, hypertension, smoking, cholesterol (LDL and HDL), or triacylglycerols.
As shown in Table 1
, higher serum concentrations of homocysteine were measured in the C. pneumoniae-seropositive group (22.3 ± 11.0 µmol/L) than in the seronegative group (17.7 ± 4.8 µmol/L). Such a difference in homocysteine concentrations between the seropositive and seronegative subjects could be found independently of the clinical manifestations of atherosclerosis, i.e., for patients suffering from coronary heart disease, from cerebrovascular occlusive disease, or from peripheral arterial occlusive disease.
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As shown in Table 1
, there were distinctly higher homocysteine concentrations for seropositive subjects in subgroups of age, renal function, smoking status, and MTHFR gene mutations. These variables influencing serum homocysteine, as well as vitamin B6, vitamin B12, and folic acid, are moreover homogeneously (not significantly different) distributed between the C. pneumoniae-seropositive and -seronegative groups. Drugs possibly influencing serum homocysteine were either well balanced between the two groups or not prescribed (antagonists of folic acid, cyclosporine, anticonvulsants).
Possible confounding effects of these variables and inflammatory markers were considered by multiple regression analysis. However, the difference in serum homocysteine observed between the C. pneumoniae-seropositive and -seronegative groups remained significant (P <0.005). The regression analysis also revealed considerable effects on serum homocysteine by age (P = 0.012), creatinine concentration (P <0.005), smoking (P = 0.023), MTHFR gene mutations (P = 0.082), and tumor necrosis factor- (P = 0.038). Vitamin B6, vitamin B12, folic acid, C-reactive protein, and interleukin 6 were only weakly associated with serum homocysteine (P >0.1) and, therefore, were excluded from analysis.
Our results indicate that C. pneumoniae seropositivity is linked with hyperhomocysteinemia in male patients with established atherosclerosis. This correlation was statistically significant no matter whether IgG (as shown above) or IgA was used as the marker for seropositivity. Understanding the relationship between the two variables requires further investigations, which are likely to be facilitated by the recent sequencing of the C. pneumoniae genome (5). Hyperhomocysteinemia is a predictor of morbidity and mortality in patients with atherosclerosis. If the association of hyperhomocysteinemia and C. pneumoniae seropositivity can be confirmed in further studies, C. pneumoniae seropositivity may be associated with impaired prognosis in these patients.
Physiologic, pathologic, genetic, and nutritional factors may increase serum homocysteine (6). Some of these variables were used as exclusion criteria (diabetes mellitus, malignant neoplasia, chronic inflammatory disorders, severe renal insufficiency). Other variables (age, renal function, smoking, MTHFR gene mutations, vitamins, concomitant medication) were found not to be responsible for the difference in homocysteine between C. pneumoniae-seropositive and -seronegative patients. This was concluded from multiple regression analysis and the observation that the possibly confounding variables were homogeneously distributed between the C. pneumoniae-seropositive and -seronegative groups. The validity of the study is further supported by the finding that distinctly higher homocysteine concentrations for seropositive subjects were measured not only in the whole study population, but also and independently in various subgroups investigated. The generally high serum concentrations of homocysteine found in this study may be explained by our selection of older patients (69.9 ± 10.1 years), with a high prevalence of smoking (>80%) and suffering from advanced atherosclerosis.
Acknowledgments
We thank Dr. G. Hinz, MEDIDATA, and Dr. P. Suter, University Hospital of Zurich, for valuable discussion. The project was financially supported by Aventis Pharma AG, Switzerland.
Footnotes
1 these authors contributed equally to this work; 
References
The following articles in journals at HighWire Press have cited this article:
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  G. Schulthess, P. Wiesli, and F. E. Maly Macrolide Treatment Does Not Influence Serum Homocysteine in Chlamydia pneumoniae-seropositive Patients Suffering from Atherosclerosis Clin. Chem., September 1, 2002; 48(9): 1631 - 1631. [Full Text] [PDF]
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 P. Wiesli, W. Czerwenka, A. Meniconi, F. E. Maly, U. Hoffmann, W. Vetter, and G. Schulthess Roxithromycin Treatment Prevents Progression of Peripheral Arterial Occlusive Disease in Chlamydia pneumoniae Seropositive Men: A Randomized, Double-Blind, Placebo-Controlled Trial Circulation, June 4, 2002; 105(22): 2646 - 2652. [Abstract] [Full Text] [PDF]
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