Association Between C-Reactive Protein, Anti-Chlamydia Pneumoniae Antibodies, and Vascular

Function in Healthy AdultsNiraj Sharma, MD, John D. Rutherford, MB, CLB, J. Thomas Grayston, MD,Louise P. King, BA, JD, Ishwarlal Jialal, MD, and Thomas C. Andrews, MD

There is growing body of evidence suggesting anassociation between serum markers of infection

and inflammation and rupture of the atheroscleroticplaque.1 However, the role of infection and inflamma-tion in early atherogenesis has not been well studied.Abnormalities of vascular function occur in patientswith risk factors for atherosclerosis before the devel-opment of obstructive disease.2 This pilot study inves-tigates a potential association between elevated levelsof C-reactive protein (CRP), anti-Chlamydia pneu-moniae, immunoglobulin G (IgG), and A (IgA) anti-bodies, and functional abnormalities of the vascula-ture in subjects at otherwise low risk for atheroscle-rosis.

• • •We recruited 98 healthy men and women, aged 18

to 49 years, by advertisement. Subjects with a historyof diabetes mellitus, dyslipidemia, hypertension, renaldisease, or cardiovascular disease were excluded. Fe-male subjects were excluded if pregnant or activelytaking oral contraceptives.

Women were studied during active menses. Base-line demographic data were recorded and blood wasobtained after an overnight fast. Measurements ofbrachial artery diameter and blood flow were obtainedwith high-resolution ultrasonography under baselineconditions, during hyperemia, and after sublingualnitroglycerin administration. The details of methodsof brachial ultrasonography used in our laboratory arepublished elsewhere.3–5 Briefly, subjects were studiedafter an overnight fast. Images were obtained with a5/7.5-MHz linear transducer and an Acuson 128XPultrasound system with vascular imaging software(Acuson, Mountain View, California). Baseline 2-di-mensional longitudinal images of the left brachialartery were obtained at a location between 2 and 10cm above the antecubital fossa. Baseline flow wasestimated with continuous-wave Doppler by recordingthe time-velocity integral sampling at midvessel. Thearm was rendered ischemic by inflating a blood pres-sure cuff placed distal to the imaging transducer tosuprasystolic pressure for 5 minutes. After the cuff

was rapidly deflated, hyperemic flow was assessedwith continuous-wave Doppler, and flow-mediatedvasodilatation was assessed by imaging the brachialartery continuously with the transducer maintained atits original location. Subjects rested quietly for 5 min-utes, and scans were repeated 3 minutes after sublin-gual administration of nitroglycerin spray (0.4 mg).Images corresponding to the peak of the electrocar-diographic R wave was digitized and all images werestored on S-VHS tapes and analyzed on a Macintosh(7500/100) computer system (Palo Alto, California).The average segment diameter was determined foreach image using customized NIH Image software(Joe Polak, Brigham and Women’s Hospital, Boston,Massachusetts), and the percent change in diameterwas calculated.

Antichlamydial IgG and IgA were measured usingmicroimmunofluorescence by the laboratory of DrThomas Grayston at the University of Washington atSeattle. CRP was determined on the Beckman Array360 (Abbott Laboratories, Abbott Park, Illinois) byrate nephelometry, which measures the rate of lightscatter formation resulting from an antigen-antibodyreaction. A single point calibrator was used to cali-brate the instrument. The coefficient of variation forintra- and interassay precision was,3.0% and thelinear range was 0.4 to 9.0 mg/dl. CRP also wasmeasured by enzyme immunoassay using theIMxUltra-CRP assay (Abbott Laboratories), a 2-stepsandwich microparticle enzyme immunoassay. Theassay utilizes mouse monoclonal anti-CRP–coatedmicroparticles that bind to CRP in the sample. Sub-sequent incubations with a mouse monoclonal anti-CRP alkaline phospatase conjugate and substrate re-sults in a fluorescent product that is measured quanti-tively. The coefficient of variation for intra- andinterassay precision was,5.0% and the linear rangewas 0.05 to 6.0 mg/dl. Lipoprotein(a) was measuredusing a sensitive enzyme-linked immunosorbent assay(Dynatech MRX Elisa Plate, Northwest Lipid Re-search Laboratories, University of Washington at Se-attle, Seattle, Washington). Total homocysteine levelswere measured using a modification of a fully auto-mated high-performance liquid chromatography assay(Dionex GP40 Gradient Pump, Sunnyvale, California;AS3500 Autosampler, Jasco FP-920 Fluorescence De-tector, Easton, Maryland). Lipid profile was measuredby b-estimation as described previously (Paramax Rx,Dade Internatoinal Inc, Miami, Florida).6

Statistical analyses were performed using SAS ver-sion 6.12 (SAS Institute, Cary, North Carolina). Alldata are presented as mean6 SD. Analysis of vari-

From the University of Texas Southwestern Medical School, Dallas,Texas; and University of Washington at Seattle, Seattle, Washington.This study was supported by grants from the American Heart Associ-ation, Texas Affiliate, and the Donald W. Reynolds Foundation, Dal-las, Texas. Dr. Andrews’ address is: Donald W. Reynolds ClinicalResearch Center, The University of Texas Southwestern Medical Centerat Dallas, 5323 Harry Hines Boulevard, H8.116, Dallas, Texas75390-9034. E-mail: Tom.Andrews@email.swmed.edu. Manuscriptreceived June 27, 2000; revised manuscript received and acceptedJuly 17, 2000.

119©2001 by Excerpta Medica, Inc. All rights reserved. 0002-9149/01/$–see front matterThe American Journal of Cardiology Vol. 87 January 1, 2001 PII S0002-9149(00)01287-X

ance and Spearman correlation coefficients were cal-culated for known predictors of endothelial functionas well as for CRP and anti-C. pneumoniaeIgG andIgA antibodies. Student’st test (pooled variances) wasalso used to compare flow and nitroglycerin-mediatedvasodilation with other variables.

Baseline characteristics of the study group are de-picted in Table 1. Fifty-six subjects (57%) were menand there was 1 active smoker. Table 2 lists the resultsof serum markers of infection and/or inflammation.Sixty-five subjects (66%) had IgG antibodies toC.pneumoniaeat a titer $1:2, and of these, 14 hadmeasurable IgA antibodies as well. Forty percent ofsubjects positive for IgG had detectable levels of CRPby the nephelometric immunoassay, as did 36% ofsubjects with detectable antichlamydial IgA. Amongsubjects with detectable CRP, 79% had detectableantichlamydial IgG and 15% had detectable IgA anti-bodies.

The relation between endothelial-dependent (flow-mediated) and independent (nitroglycerin-mediated)vasodilation and the markers of inflammation are dis-played in Table 3. No significant difference in endo-thelium-independent or dependent vasodilation wasobserved in subjects with and without elevated IgG

levels. Elevation of IgA was associated with 27% lessflow-mediated vasodilation (p5 0.37) and 12% lessnitroglycerin-mediated vasodilation (p5 0.42). CRPelevation was associated with a 23% reduction inflow-mediated vasodilation (p5 0.3) and a 30.7%reduction in nitroglycerin-mediated vasodilation (p50.07). Subjects with detectable antichlamydial IgA orCRP had 31% less flow-mediated vasodilation (p50.11) and 21% less nitroglycerin-mediated vasodila-tion (p 5 0.039). The correlation between absolutelevels of CRP measured by ultrasensitive assay andflow- or nitroglycerin-mediated vasodilation was lessrobust than with CRP measured with nephelometry.There was no correlation between levels of lipopro-tein(a) homocysteine and low-density lipoprotein cho-lesterol, although the mean value for each of thesevariables was below the population mean.

• • •We performed this pilot study to determine

whether infection withC. pneumoniaeand/or evi-dence of inflammation is associated with endothelial

dysfunction. We chose healthy sub-jects without other risk factors forcoronary artery disease to minimizepotential confounders. Certainly, in-fection and inflammation may actsynergistically with other risk factorsto initiate or accelerate atherogene-sis. Thus, in a population with coro-nary risk factors, the effect of raisedCRP and or IgA may be accordinglylarger. In our study, most subjects(66%) had prior infection withC.pneumoniae, and there was no rela-tion between the presence or level ofIgG antibodies and endothelial-de-pendent or independent vasodilation

of the brachial artery. However, antichlamydial IgAantibodies decrease more rapidly after acute infection,and elevated levels may suggest either relatively re-cent acute infection or chronic, persistent infection.7

Our data demonstrate consistent trends in reducedflow-mediated and nitroglycerin-induced vasodilationin subjects with IgA antibodies toChlamydiaor evi-dence of inflammation reflected by detectable levels ofCRP by the nephelometric immunoassay. In 1 sub-group, this trend reached statistical significance: insubjects with both detectable IgA and CRP, endothe-lium-dependent vasodilation was 31% lower (p,0.10) and endothelial-independent vasodilation 21%lower (p ,0.05) than in subjects without detectablelevels of either marker. There are limitations in usingantibodies to detect persistentC. pneumoniaeinfec-tion, although the association of CRP, IgA antibodies,and vascular dysfunction is intriguing. Other tech-niques to identify persistent infection, such as poly-merase chain reaction forC. pneumoniaedeoxyribo-nucleic acid, may prove more useful in future studies.8

If infection and inflammation are important in earlyatherogenesis as our preliminary results suggest, vac-cines or antimicrobial therapy potentially could be

TABLE 3 Relation Between anti-Chlamydia pneumoniae IgA, C-Reactive Protein,and Flow- and Nitroglycerin-Mediated Vasodilation

Present Absent p Value

Anti-Chlamydia pneumoniae IgAFlow-mediated vasodilation (%) 3.5 6 6.1 4.8 6 5.0 0.37Nitroglycerin-induced vasodilation (%) 16.7 6 5.6 19.0 6 10.8 0.42

C-reactive proteinFlow-mediated vasodilation (%) 4.0 6 5.6 5.1 6 4.9 0.30Nitroglycerin-mediated vasodilation (%) 16.2 6 9.7 23.4 6 22.0 0.07

Anti-Chlamydia IgA or C-reactive proteinFlow-mediated vasodilation (%) 3.7 6 5.5 5.3 6 4.7 0.11Nitroglycerin-induced vasodilation (%) 16.2 6 9.1 20.5 6 10.7 0.039

TABLE 1 Demographics and Baseline Variables

Variable Mean 6 SD

Age (yrs) 29 6 7Body mass index (kg/m2) 25 6 4Systolic blood pressure (mm Hg) 115 6 11Diastolic blood pressure (mm Hg) 74 6 9Total cholesterol (mg/dl) 171 6 32Low-density lipoprotein cholesterol (mg/dl) 104 6 27High-density lipoprotein cholesterol (mg/dl) 46 6 13Triglycerides (mg/dl) 110 6 74Lipoprotein(a) (mg/dl) 23 6 40Homocysteine (mmol/L) 8.5 6 5.2

TABLE 2 Markers of Infection and Inflammation

Marker Mean 6 SD Median Range

IgG titers 1:48 0–1:1,024IgA titers 0 0–1:128CRP (mg/dl)

Nephelometric 2.8 6 1.7 ,0.4 ,0.4–16.5Ultrasensitive 6.35 6 2.86 1.031 ,0.08–41.09

120 THE AMERICAN JOURNAL OF CARDIOLOGYT VOL. 87 JANUARY 1, 2001

used to treat or prevent the clinical manifestations ofthis disease.

Abnormalities of vascular function occur in pa-tients with risk factors for atherosclerosis beforethe development of obstructive disease. Our pilotdata suggest that elevated serum markers of infec-tion and/or inflammation are associated with func-tional abnormalities of the vasculature in subjectsat otherwise low risk for atherosclerosis.

1. Ridker P, Cushman M, Stampfer M, Tracy R, Hennekens C. Inflammation,aspirin, and the risk of cardiovascular disease in apparently healthy men.N EnglJ Med1997;336:973–979.2. Celermajer D, Sorensen K, Gooch V, Spiegelhalter D, Miller O, Sullivan I,

Lloyd J, Deanfield J. Non-invasive detection of endothelial dysfunction in chil-dren and adults at risk of atherosclerosis.Lancet1992;340:1111–1115.3. Greiner D, Personius B, Andrews T. Effects of withdrawal of chronic estrogentherapy on brachial artery vasoreactivity in women with coronary artery disease.Am J Cardiol1999;83:247–249.4. English J, Green G, Jacobs L, Andrews T. Effect of the menstrual cycle onendothelium-dependent vasodilation of the brachial artery.Am J Cardiol1998;82:256–258.5. Andrews T, Whitney E, Green G, Kalenian R, Personius B. Effect of gemfi-brozil 6 niacin6 cholestyramine on endothelial function in patients with serumlow-density lipoprotein levels,160 mg/dl and high-density lipoprotein choles-terol levels,40 mg/dl.Am J Cardiol1997;80:831–835.6. Hirany S, Li D, Jialal I. A more valid measurement of low-density lipoproteincholesterol in diabetic patients.Am J Med1997;102:48–53.7. Saikku P, Leinonen M, Tenkanen L, Linnanmaki E, Ekman MR, Manninen V,Manttari M, Frick MH, Huttunen JK. Chronic chlamydia pneumoniae infection asa risk factor for coronary heart disease in the Helsinki Heart Study.Ann InternMed 1992;116:273–278.8. Moazed T, Kuo C, Grayston J, Campbell L. Evidence of systemic dissemina-tion of Chlamydia pneumoniae via macrophages in the mouse.J Infect Dis1998;177:1322–1325.

A Noninvasive Measurement of Reactive HyperemiaThat Can Be Used to Assess Resistance Artery

Endothelial Function in HumansYukihito Higashi, MD, PhD, Shota Sasaki, MD, Keigo Nakagawa, MD,

Hideo Matsuura, MD, PhD, Goro Kajiyama, MD, PhD, and Tetsuya Oshima, MD, PhD

Several methods have been used to assess endothe-lial function in humans.1–5 The intra-arterial infu-

sion of nitric oxide agonists, such as acetylcholine,methacholine, and bradykinin, and nitric oxide antag-onists demonstrate the important role of nitric oxiderelease. Although this method has been used to inves-tigate endothelial function in humans, it is not avail-able for routine measurement because it is an invasivetechnique, requiring catheter insertion into the bra-chial or femoral artery and takes several hours toperform. A noninvasive method of measuring reactivehyperemia (RH) has recently been demonstrated to beuseful in assessing resistance vessel endothelial func-tion.6 Several investigators, including our group, haveshown that blood flow response during RH is impairedin patients with essential hypertension.7–9 However,there is little information regarding the validity of thisnoninvasive method in the assessment of resistancevessel endothelial function. In the present study, weevaluated the effects of RH and intra-arterial acetyl-

choline infusion on forearm blood flow (FBF) in nor-mal subjects and patients with essential hypertensionto determine the correlation between the 2 methods. Theresponses of FBF to RH and acetylcholine infusion inthe absence and presence of the nitric oxide synthaseinhibitor NG-monomethyl-L-arginine (L-NMMA) werecompared.

• • •The study group consisted of 47 Japanese patients

with essential hypertension (29 men and 18 women;mean age 526 9 years; duration of hypertension: 966 years) with no objective signs of hypertensive end-organ damage and 39 normotensive subjects (25 menand 14 women; mean age 486 12 years). Hyperten-sion was defined as a systolic blood pressure.160mm Hg and/or a diastolic blood pressure.95 mm Hg,determined on$3 different examinations with thepatient in a sitting position. Patients with secondaryforms of hypertension, as suggested by clinical andbiochemical examination, were excluded from thestudy. Sixteen of 47 patients with hypertension wereon antihypertensive drugs (calcium antagonists [n511], angiotensin-converting enzyme inhibitors [n55], diuretic agents [n5 2], andb blockers [n5 2]).No antihypertensive drugs were taken for$2 weeksbefore the study. Normotension was defined as a sys-tolic blood pressure of,140 mm Hg and a diastolicblood pressure of,80 mm Hg. Normotensive sub-jects had no history of serious disease and had takenno medication for$4 weeks before the study. Sub-jects with a history of cardiovascular or cerebrovas-cular disease, diabetes mellitus, or hepatic or renaldisease were excluded from the study. The study pro-tocol was approved by the ethics committee of Hiro-

From the First Department of Internal Medicine and the Department ofClinical Laboratory Medicine, Hiroshima University School of Medi-cine, Hiroshima, Japan. This study was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Scienceand Culture of Japan (to T. Oshima) and Japan Heart Foundation Grantfor Research on Hypertension and Metabolism, Tokyo; a Grant forResearch Foundation for Community Medicine, Tokyo; a Grant forResearch on Autonomic Nervous System and Hypertension fromKimura Memorial Heart Foundation/Pfizer Pharmaceuticals Inc., Ku-rume; and Foundation for Total Health Promotion (to Y. Higashi),Tokyo, Japan. Dr. Higashi’s address is: Hiroshima University School ofMedicine, First Department of Internal Medicine, 1-2-3 Kasumi, Mi-nami-ku, Hiroshima 734-8551, Japan. E-mail: yhigashi@mcai.med.hiroshima-u.ac.jp. Manuscript received May 4, 2000; revised manu-script received and accepted July 11, 2000.

121©2001 by Excerpta Medica, Inc. All rights reserved. 0002-9149/01/$–see front matterThe American Journal of Cardiology Vol. 87 January 1, 2001 PII S0002-9149(00)01299-1