Jessie W. Shih

Prospective Study of C-Reactive Protein and the Risk of Future Cardiovascular Events Among Apparently Healthy Women

BACKGROUND C-reactive protein (CRP) predicts risk of myocardial infarction (MI) and stroke among apparently healthy men, but in women, virtually no data are available. METHODS AND RESULTS CRP was measured in baseline blood samples from 122 apparently healthy participants in the Womens Health Study who subsequently suffered a first cardiovascular event and from 244 age- and smoking-matched control subjects who remained free of cardiovascular disease during a 3-year follow-up period. Women who developed cardiovascular events had higher baseline CRP levels than control subjects (P=0.0001), such that those with the highest levels at baseline had a 5-fold increase in risk of any vascular event (RR=4.8; 95% CI, 2.3 to 10.1; P=0.0001) and a 7-fold increase in risk of MI or stroke (RR=7.3; 95% CI, 2.7 to 19.9; P=0.0001). Risk estimates were independent of other risk factors, and prediction models that included CRP provided a better method to predict risk than models that excluded CRP (all P values <0.01). In stratified analyses, CRP was a predictor among subgroups of women with low as well as high risk as defined by other cardiovascular risk factors. CONCLUSIONS In these prospective data among women, CRP is a strong independent risk factor for cardiovascular disease that adds to the predictive value of risk models based on usual factors alone. (Circulation. 1998;98:731-733.)

BASELINE IGG ANTIBODY TITERS TO CHLAMYDIA PNEUMONIAE, HELICOBACTER PYLORI, HERPES SIMPLEX VIRUS, AND CYTOMEGALOVIRUS AND THE RISK FOR CARDIOVASCULAR DISEASE IN WOMEN

Data from several studies have suggested that persons with coronary heart disease have an increased prevalence of chronic infection with such agents as Chlamydia pneumoniae, Helicobacter pylori, herpes simplex virus, and cytomegalovirus (1). In addition, it has been hypothesized that infection may be a risk factor for acute coronary events (2). However, chronic infection is also more prevalent among smokers, elderly persons, and persons of lower socioeconomic status, and persons with a history of coronary disease may be more susceptible to subsequent infection. Therefore, it is uncertain whether the associations between infection and coronary heart disease that have been observed in retrospective and cross-sectional studies were caused by confounding or represent a result of ischemic heart disease rather than a cause (3). To resolve these issues, investigators have used prospective, controlled settingsin which exposure status can be ascertained before the onset of thrombosisto evaluate the theory that previous infection is related to atherosclerosis. The few reported prospective studies of C. pneumoniae (4-7), H. pylori (8-12), herpes simplex virus (13), and cytomegalovirus (13) have not provided strong evidence of an association; however, none of these studies evaluated multiple infectious exposures simultaneously. This is a potentially important issue because it has been hypothesized that a persons total burden of pathogens may be a critical factor in determining atherogenesis (14). In addition, the available prospective data were derived from studies that predominantly or exclusively evaluated men. To further investigate the theory that previous infection is related to atherothrombosis, we measured IgG antibody titers against C. pneumoniae, H. pylori, herpes simplex virus, and cytomegalovirus in baseline blood samples obtained from a large cohort of apparently healthy postmenopausal women who were followed prospectively for the occurrence of first cardiovascular events. We also related these antibody titers to plasma concentrations of high-sensitivity C-reactive protein, a marker of chronic inflammation that has previously been shown to predict coronary risk in this cohort (15). Methods We performed a nested casecontrol study among participants in the Womens Health Study, an ongoing primary prevention trial that enrolled 39 876 postmenopausal female health professionals with no history of myocardial infarction, stroke, or transient ischemic attack (16). Of women enrolled in the study, 28 311 (71%) provided baseline blood samples, which were frozen in liquid nitrogen until analysis. Case-patients were initially healthy Womens Health Study participants who provided a baseline blood sample and who subsequently reported a first cardiovascular event (myocardial infarction, stroke, cardiovascular death, or coronary revascularization procedure) during follow-up. Reported myocardial infarction was confirmed if review of hospital records met World Health Organization criteria and if the event was associated with characteristic electrocardiographic changes or elevated levels of cardiac enzymes. Reported stroke was confirmed if records showed a new neurologic deficit persisting for more than 24 hours; such records almost always included evidence from computed tomography or magnetic resonance imaging. Reported coronary angioplasty or coronary bypass surgery was confirmed by record review. Cardiovascular death was confirmed by autopsy reports, death certificates, and circumstances at the time of death. Controls were randomly selected from the pool of initially healthy Womens Health Study participants who remained free of cardiovascular disease during study follow-up and also provided a baseline blood sample. Two controls, matched for age (1 year) and smoking status (never, past, or current), were selected for each case-patient. Baseline plasma samples from case-patients and controls were thawed and assayed for IgG antibody titers against C. pneumoniae by using microimmunofluorescence techniques (6, 17). Similarly, enzyme-linked immunosorbent assays were used to qualitatively detect IgG antibodies to herpes simplex virus (Wampole Laboratories, Cranbury, New Jersey), cytomegalovirus (Gull Laboratories, Salt Lake City, Utah), and H. pylori (Wampole Laboratories) in baseline plasma samples. For each variable, samples from a case-patient and two matched controls were assayed as a group; samples from the case-patient were positioned randomly within the group to reduce interassay variability and to avoid systematic bias. All laboratory investigators were unaware of case-patient or control status at the time of IgG analysis. C-reactive protein levels were evaluated by using a high-sensitivity assay (Abbott Laboratories, Abbott Park, Illinois), as described elsewhere (15). We used conditional logistic regression analyses to test for evidence of an association between the presence of IgG antibodies at baseline and subsequent risk for cardiovascular events. A priori, we chose to evaluate the association between C. pneumoniae and subsequent risk across a series of IgG antibody titers (range,>1:16 to>1:128). The presence or absence of IgG seropositivity for herpes simplex virus, cytomegalovirus, and H. pylori was determined according to cut-points established by the assay manufacturers. Adjusted estimates of risk were computed after we controlled for baseline differences between case-patients and controls. To evaluate the theory that total infectious burden rather than any single IgG titer may be associated with risk, we further classified study participants as having zero, one, two, three, or four positive antibody titers. Because the number of study participants with zero positive titers at baseline was small (n=15), data from these participants and from participants with one positive titer were combined for analysis. Where applicable, tests for trend were used to evaluate evidence of increasing risk across increasing antibody titers (18). We also compared the distribution of C-reactive protein values for participants with two or more positive IgG antibody titers with the distribution of values for participants with zero or one positive IgG antibody titer. All P values are two-tailed. Results Among 122 case-patients, 85 myocardial infarctions or strokes occurred and 37 coronary revascularizations were performed. As is expected in a study of incident coronary events, case-patients were more likely than controls to have a history of hyperlipidemia (45.9% compared with 28.3%; P=0.001), hypertension (55.5% compared with 31.3%; P=0.001), and diabetes (9.8% compared with 2.1%; P=0.001) and to have a family history of premature coronary disease (21.3% compared with 12.7%; P=0.04). Case-patients also had a greater mean body mass index than controls (27.1 compared with 26.0 kg/m2; P=0.05). Because of matching, mean age (59.3 8.3 years) and smoking status (27.9% of participants currently smoked, 42.6% had never smoked, and 29.5% had smoked in the past) were identical in the case-patient and control groups. Exposure rates among controls were similar to those reported in previous studies; for example, 60% of controls had C. pneumoniae titers greater than 1:8 (1, 2). However, as shown in Table 1, the proportion of study participants with positive IgG titers was similar regardless of case-patient or control status. For example, the crude rate ratios for future cardiovascular events in women with C. pneumoniae titers 1:16, 1:32, 1:64, and 1:128 were 1.1, 1.1, 1.1, and 1.0, respectively (P>0.2 overall) (Table 1). Similarly, the crude rate ratios for future cardiovascular events associated with baseline seropositivity to H. pylori, herpes simplex virus, and cytomegalovirus were 0.9, 1.2, and 0.9, respectively (P>0.2 overall). As shown in Figure 1, these point estimates did not change after adjustment for baseline differences in hyperlipidemia, hypertension, exercise frequency, body mass index, diabetes, and family history of premature coronary artery disease. Table 1. Rate Ratios for Future Cardiovascular Events among Apparently Healthy Women, according to Baseline IgG Antibody Status Figure 1. Adjusted rate ratios for future cardiovascular events among apparently healthy women, according to the presence of baseline IgG antibody titers against Chlamydia pneumoniae , herpes simplex virus, cytomegalovirus, and Helicobacter pylori ( top ) and the number of positive baseline IgG titers present ( bottom ). To evaluate the theory that total pathogen burden might be associated with increased risk, we stratified patients into four groups according to the total number of positive titers observed in a given case-patient or control. As shown in Table 2, the rate ratios for future cardiovascular events in women with zero or one, two, three, or four positive IgG titers were 0.9, 1.1, 1.0, and 1.0, respectively (P>0.2 overall). Similarly, in analyses that evaluated evidence of trend across these four groups, little evidence supported an association (P>0.2 for trend). As shown in Figure 1, adjustment for baseline differences between case-patients and controls had little or no effect on these results. Table 2. Rate Ratios for Future Cardiovascular Events among Apparently Healthy Women, according to the Total Number of IgG Antibody Titers Present in a Given Study Participant Previously obtained data from this cohort indicate that median C-reactive protein levels are significantly higher in participants who subsequently reported coronary events than in controls (6.45 compared with 3.75 mg/L; P<0.001); this suggests that chronic low-grade inflammation is a marker of risk in this group of women (15). However, when we compared participants with and those without positive IgG antibody titers, the distribution of C-reactive protein levels was similar in isolated analyses limited to each pathogen, in analyses evaluating total pathogen burden (Figure 2), and in analy

Effect of Collection Tube Type and Preanalytical Handling on Myeloperoxidase Concentrations

BACKGROUND Myeloperoxidase (MPO) has shown potential as a marker for cardiovascular disease. Limited studies have been published with a variety of sample types, resulting in a wide range of MPO values. Little is known or understood about the impact of collection tube type and preanalytical handling of specimens for MPO determination. METHOD MPO concentration was determined by use of the ARCHITECT(R) MPO research use assay, which is currently under development. Samples were collected into multiple anticoagulant collection tubes from donors and patients presenting to the emergency department with symptoms of acute coronary syndromes. Whole blood was stored on ice or at room temperature for predetermined time periods. We also evaluated serum and plasma after centrifugation followed by storage at room temperature, 2-8 degrees C, and below -10 degrees C. RESULTS Baseline sample concentrations were dependent on collection tube type as well as handling conditions. MPO concentrations were consistently higher in samples collected in serum and heparin plasma tubes than in samples in EDTA or citrate tubes. Spike recovery was acceptable in all sera and plasma tested, indicating that the increased MPO concentrations were not due directly to an anticoagulant interference. CONCLUSIONS The collection tube type and preanalytical handling are critical for accurate and consistent MPO measurement. The preferred anticoagulant and tubes are the EDTA or EDTA plasma preparation tube. MPO concentrations in samples collected in these tubes are stable before centrifugation as whole blood as well as plasma after processing.

Plasma Homocysteine Concentration, Statin Therapy, and the Risk of First Acute Coronary Events

Background—Elevated homocysteine levels are associated with increased coronary risk, and it has been suggested that homocysteine screening may provide a method to identify high-risk patients for aggressive primary prevention. Methods and Results—Homocysteine was measured at baseline and after 1 year among 5569 participants in the Air Force/Texas Coronary Atherosclerosis Prevention Study (AFCAPS/TexCAPS), a randomized trial of lovastatin in the primary prevention of acute coronary events. The effects of homocysteine, LDL cholesterol, and lovastatin on risk were assessed over 5.2 years of trial follow-up. Median baseline homocysteine levels were significantly higher among study participants who subsequently had acute coronary events compared with those who did not (12.1 versus 10.9 &mgr;mol/L, P <0.001). The relative risks of future events from lowest (referent) to highest quartile of homocysteine were 1.0, 1.6, 1.6, and 2.2 (P <0.001). These effects were similar among those allocated to lovastatin and those allocated to placebo and were modestly attenuated after adjustment for other traditional risk factors. As predicted, the subgroup of participants with elevated LDL cholesterol and elevated homocysteine levels were at high risk and benefited greatly from statin therapy (relative risk, 0.46; 95% CI, 0.29 to 0.75; number needed to treat=26). However, in contrast to findings in this trial for C-reactive protein, homocysteine evaluation did not help to define low LDL subgroups with different responses to lovastatin therapy. Conclusions—Although homocysteine predicted future coronary events in AFCAPS/TexCAPS, we found little evidence that homocysteine evaluation provided an improved method to target statin therapy among those with low-to-normal LDL cholesterol levels.

C-reactive protein and other markers of inflammation among patients undergoing HELP LDL apheresis

Studies reveal important prognostic relationships between C-reactive protein (CRP) and atherosclerotic complications. A prospective trial of familial hypercholesterolemic patients treated with Heparin-induced Extra-corporeal Low-Density Lipoprotein Precipitation (HELP, B. Braun Melsungen) therapy was undertaken to evaluate the short- and long-term effects on CRP. Four patients received LDL apheresis therapy on an alternate week basis for 6 months. Pre- and post-treatment serum high sensitivity (hs) CRP levels (IMx(R), Abbott Laboratories), LDL-C, triglycerides, and fibrinogen were measured. Pre- and post-treatment mean serum levels of LDL-C were 281+/-76 and 98+/-34 mg/dl; triglycerides 191+/-64 and 123+/-50 mg/dl; fibrinogen 332+/-46 and 117+/-31 mg/dl, respectively. Before and after apheresis mean serum levels of hsCRP were 8.99+/-7.88 and 3.15+/-3.16 mg/ml, respectively, representing a 65% decrease. After 6 months of therapy, pre-treatment hsCRP showed an overall mean level decrease of 49%. Preliminary results indicate that LDL apheresis results in a rapid and long-term decrease of serum hsCRP levels.

Cardiac Troponin Scorecard

The recent opinion paper by Apple (1) on a cardiac troponin assay scorecard has enlarged the conundrum on whether cardiac troponin assays are clinically usable or not. Apple has proposed a scorecard that evaluates cardiac troponin assays according to the total imprecision at the 99th percentile. The cited data include the concentrations at the 10% CV and whether the value is greater or less than the cited 99th-percentile value. No data for the 20% CV are presented, however, and all of the cited values are derived from separate independent studies. Values for the 99th percentile of a healthy population have been reported to show considerable variation between assays and even within an assay (2), due to such factors as the reference population chosen, the skewness of the distribution, specimen type, exclusion criteria, and sample size. In addition, the selection of the zero-concentration calibrator …

Potential Interference by Antineutrophil Cytoplasmic Autoantibodies in Myeloperoxidase Immunoassays

Myeloperoxidase (MPO)1 has been identified as a potential marker of cardiovascular disease (1). As the clinical utility of new biomarkers is identified and commercial assays are developed, limitations on their use need to be identified and reported. Antineutrophil cytoplasmic autoantibodies (ANCAs) are known to exist in patients with diseases characterized by primary systematic vasculitis, such as Wegener granulomatosis, microscopic polyangiitis, and Churg– Strauss syndrome, and in those with idiopathic pauci-immune necrotizing crescentic glomerulonephritis, systemic lupus erythematosus, and rheumatoid arthritis (2). In particular, the immunofluorescence- staining pattern for perinuclear ANCAs (pANCAs) is associated with anti-MPO autoantibodies. pANCAs are most prevalent in microscopic polyangiitis (40%–80%), followed by Churg–Strauss syndrome (20%–30%) and Wegener granulomatosis (5%–20%). Autoantibodies can interfere with assays when the autoantibodies bind to analyte epitopes that are similar to those of the monoclonal antibodies used in the immunoassay. Anti-MPO autoantibodies are likely to bind to MPO in the sample and block or partially mask the epitopes necessary for binding to the antibodies used to capture and detect MPO. We evaluated the effect of MPO autoantibodies on the performance of the Cardio MPO™ immunoassay (PrognostiX), which has been cleared by the US Food and Drug Administration. We similarly evaluated an immunoassay under development for the Abbott ARCHITECT® instrument (Abbott …