March 2011
Feature Story

William Check, PhD

Inflammation is pivotal in all phases of atherosclerosis, from the fatty streak to plaque rupture, and thus biomarkers of inflammation that predict coronary artery disease and its sequelae are much studied and talked about. In a session at the 2010 meeting of the American Society for Clinical Pathology, “Update of Cardiovascular Disease and Markers,” four such analytes were the focus: C-reactive protein, myeloperoxidase, adiponectin, and non-HDL-cholesterol.

One of the speakers, Ishwarlal Jialal, MD, PhD, FRCPath (London), DABCC, called C-reactive protein “the best biomarker we have for quantifying inflammation,” one that has been shown repeatedly to predict CVD in healthy people and mortality in those with myocardial infarction and coronary heart disease. Dr. Jialal, who is Robert E. Stowell endowed chair in experimental pathology and director of the laboratory for atherosclerosis and metabolic research in the Department of Pathology and Laboratory Medicine at the University of California, Davis, Medical Center, says the Canadian guidelines for CVD prevention, which came out at the end of 2010, endorsed the lab test for high-sensitivity CRP to screen men over 50 and women over 60 for CHD. The Centers for Disease Control and Prevention has similar recommendations, he says, and the American Heart Association and the National Heart, Lung and Blood Institute previously endorsed CRP for risk stratification.

Dr. Jialal, who is also professor of internal medicine (endocrinology, diabetes, and metabolism), extended his consideration of risk assessment to the metabolic syndrome, which has become epidemic in the United States. One of his suggestions: “Testing for microalbuminuria and reporting non-HDL-cholesterol should be routine, and maybe the new guidelines will recommend apolipoprotein B measurement also.”

In the other talk of the session, Martin Kroll, MD, chief of the Department of Laboratory Medicine at Boston Medical Center, discussed non-HDL-C, as well as apolipo­protein B; and lipo­protein-associated phospholipase A2 (Lp-PLA₂), which signals vulnerable (“rupture-prone”) plaque. (Dr. Kroll was ill at the time of the meeting, so Dr. Jialal’s colleague, Sridevi Devaraj, PhD, associate professor of pathology and laboratory medicine and director of the clinical laboratory in the Clinical and Translational Science Center Clinical Research Center, UC Davis Medical Center, delivered his talk.)

Dr. Kroll also discussed the use of brain natriuretic peptide and NT-proBNP in patients with renal dysfunction. “You can’t look at heart function and renal function independently,” he told CAP TODAY in a recent interview. “These are two variables that have mutual effects on each other.”

Discussing with CAP TODAY the advantage of non-HDL-C, Dr. Kroll said, “In my experience, low-density lipoprotein cholesterol is frequently low or negative because of high triglycerides.” At one hospital he asked to be notified when outpatients had an LDL-C less than 70 mg/dL. They all had high triglycerides because they were not fasting. “This messes up the calculations,” Dr. Kroll says. “Non-HDL-C is not confounded by that.”

Non-HDL-C was introduced in the third report of the National Cholesterol Education Program’s Adult Treatment Panel III as a secondary therapeutic target when serum triglyceride, or TG, is 200 mg/dL or greater. Non-HDL-C includes all atherogenic particles: LDL, very-low-density lipoprotein, intermediate-density lipoprotein, and chylomicrons.

In a large meta-analysis by the Emerging Risk Factors Collaboration, non-HDL-C had a 1.5 hazard ratio for CVD events in 300,000 subjects with no CVD history (JAMA. 2009; 302:1993–2000). Similarly, among 18,000 men who entered the Health Professionals Follow-Up Study without signs of CVD, both non-HDL-C and apoB were important and significant predictors of CVD outcome. When evaluated by quintiles, non-HDL-C and apoB more strongly predicted CVD risk than did LDL-C (Pischon T, et al. Circulation. 2005; 112:3375–3383). Another group found that non-HDL-C and apoB were both superior to LDL-C for assessing risk during statin treatment (Kastelein JJ, et al. Circulation. 2008;117:3002–3009).

Pischon, et al and Kastelein, et al recommended substituting either non-HDL-C or apoB for LDL-C in assessing risk. “You can choose between them,” Dr. Kroll says. “They are basically measuring the same thing.” One disadvantage of apoB is that the test is not so common, while non-HDL-C can be derived from a simple lipid profile. “Non-HDL-C is actually one less measurement than a current lipid panel,” Dr. Kroll points out.

However, he adds, “We don’t know how this is going to turn out.” One obstacle is physician expectation that LDL-C will appear on laboratory reports. “We have to convince people to drop LDL-C and to support non-HDL-C or apoB,” Dr. Kroll says. “That’s sort of where the field is moving.” LDL-C has not yet been dropped at Boston Medical Center. “It’s a big challenge to re-educate the clinicians,” he explains. “It is much easier to do a scientific study than to change behavior.”

Like Dr. Kroll, Dr. Jialal advocates use of non-HDL-C, saying, “This is a test that labs should be reporting.” It is a simple calculation—total cholesterol minus HDL cholesterol. “Converting non-HDL-C to a therapeutic target is easy—add 30 to HDL,” Dr. Jialal says. “We report it. The data are all there, and there is no extra expense.”

Alan Wu, PhD, not a presenter in the ASCP session, agrees there is “a sort of Mafia” that believes the world revolves around LDL-C.

“A lot of people feel that non-HDL cholesterol would be a better marker,” he told CAP TODAY. ApoB is superior to LDL-C for assessing cardiac risk, he says, but the National Cholesterol Education Program has not adopted it. “They are sort of stuck on LDL-C as the primary risk assessment marker,” says Dr. Wu, professor of laboratory medicine at the University of California, San Francisco, and section chief of clinical chemistry and toxicology, San Francisco General Hospital. However, standardization is an important issue. “NCEP has done a wonderful job of making that happen [with LDL-C and HDL-C],” he says. “Where we stand with apoB, until it is standardized, it is a little unrealistic to expect it to be widely adopted, even though the data might be better.”

Another marker that predicts CVD events is Lp-PLA₂, which is responsible for releasing LDL oxidation products into the artery wall. In more than 25 studies, elevated values of Lp-PLA₂ were associated with doubling risk for CVD, independently of CRP and classic risk factors such as triglycerides and HDL-C. “We can use Lp-PLA₂ as a surrogate to say a patient has a higher degree of unstable plaque,” Dr. Kroll says. In the West of Scotland Prevention Study, the relationship between Lp-PLA₂ level and CVD events was highly significant and graded by quintile (Packard CJ, et al. N Engl J Med. 2000;343:1148–1155).

A recent consensus panel recommended use of Lp-PLA₂ as an adjunct to traditional risk assessment in patients with moderate and high 10-year CVD risk (Davidson MH, et al. Am J Cardiol. 2008;101:51F–57F). Elevated Lp-PLA₂ raises the CVD risk by one category and lowers the LDL-C target by 30 mg/dL.

Natriuretic peptides—BNP and NT-proBNP—are established markers, but Dr. Kroll advises they be used with more care. Natriuretic peptides are related to the degree of heart failure and renal function; they predict systolic and diastolic failure, hospital readmission, and acute coronary syndromes. The two analytes are similar in diagnostic utility.

Many fine assays for natriuretic peptides are available, and all have minor problems, in Dr. Kroll’s view. “This is an area with lots of controversy. Different manufacturers advocate different forms of the peptide. It was said that proBNP is no good because it is sensitive to renal function. In fact, both BNP and NT-proBNP are sensitive to renal function.” The sensitivity of this analyte to renal function is magnified in conjunction with heart dysfunction. “If there is any degree of heart failure, renal function can have a greater impact,” Dr. Kroll says. The point is to pay attention to both cardiac and renal function when interpreting results for any form of natriuretic peptide.

It has long been known that total cholesterol cannot explain all cases of coronary heart disease, Dr. Jia­lal noted. Many years ago a 26-year followup of the Framingham Heart Study showed that 50 percent of coronary heart disease occurs in people with below average total cholesterol (Castelli WP. Atherosclerosis. 1996;124 [suppl]: 51–59).

Many mediators affect the progress of atherosclerosis, among them vascular cell adhesion molecules and monocyte chemotactic protein. These cytokines attract inflammatory cells from the blood into a damaged artery. Interleukins are also involved. Interleukin 6 (IL-6) exacerbates atherosclerosis in mice, and lack of IL-1beta decreases its severity. Lack of IL-10, on the other hand, increases the severity of atherosclerosis. “Interleukins are a homeostatic system,” Dr. Jialal says. “Interleukins 1 and 6 are proinflammatory while interleukin 10 is antiinflammatory.” The CD40-CD40 ligand system furthers inflammation in atheromas. Work on all these pro- and antiinflammatory substances supports the pivotal role of inflammation in atherosclerosis.

“There is a conspiracy of cells to enhance atherosclerosis by the proinflammatory pathway,” Dr. Jialal says. Endothelial cells of the atheromatous vessel act first, followed by recruited macrophages, then T-lymphocytes.

In addition to in vitro and animal studies, clinical research supported the need for new explanations for CVD. A review of four studies of angiography in the infarct-related artery showed that the extent of stenosis prior to plaque rupture was often insufficient to explain the CVD event (Falk E, et al. Circulation. 1995;92: 657–671). In fact, in these four studies overall, 68 percent of those with MI had less than 50 percent stenosis in the infarct-related artery. “When I was a medical student, they taught us that you had to occlude the whole vessel to have a heart attack,” Dr. Jialal says.

Gradually, a unifying concept emerged focusing on the central role of inflammation. First, inflammation is found in most established CVD risk factors—dyslipidemia, hypertension, diabetes, and smoking. Second, inflammation participates in all phases of atherothrombotic disease—lesion initiation, progression, and thrombotic complications.

A search for inflammatory markers as predictors of coronary artery disease risk turned up CRP as a prime candidate. Between 1997 and 2002 nearly 20 prospective studies were published showing that elevated CRP levels were associated with risk of future CAD (MI or death) in men free of clinical disease at the time of measurement. Elevated risk ranged generally between twofold and fourfold. The relation held for women as well. In fact, women with LDL-C levels less than 130 mg/dL but CRP levels greater than 3.0 mg/L had a greater chance of a cardiovascular event than women with LDL-C levels greater than 160 and CRP levels less than 1.0. Data suggest that CRP might also be able to predict risk of death following an MI (Lindahl B, et al. N Engl J Med. 2000;343: 1139–1147).

In time CRP became stratified as: <1 mg/L, low risk; 1–3, intermediate risk; 3–10, high risk.

Dr. Jialal said that, from a technical point of view, the high-sensitivity CRP assay has good specifications: It is available on many automated platforms and has good precision and sensitivity. “We have been doing this assay for eight years,” he says.

A treatment trial published in 2008 seemed to establish CRP as an efficacy marker. In JUPITER, almost 18,000 people in 26 countries were treated with the statin rosuvastatin. That they were not selected on the basis of elevated lipid levels was novel; in fact, the subjects all had LDL-C under 130 mg/dL. They were chosen for having an intermediate-risk CRP of 2.0 mg/L or above. The statin lowered both LDL-C and CRP levels. The authors concluded, “Achieved hsCRP concentrations were predictive of event rates irrespective of the lipid endpoint used, including the apolipoprotein B to apolipoprotein A1 ratio” (Ridker P, et al. N Engl J Med. 2008; 359: 2195–2207).

“JUPITER brought home that in people who were healthy and had an LDL-cholesterol level of less than 130 mg/dL, a statin reduced both LDL and CRP and cardiac events,” Dr. Jialal says. “And you couldn’t explain the benefit on LDL reduction alone.” He calls the introduction of statins “a quantum leap,” noting they have been safe and beneficial. Now their use can possibly be extended, in Dr. Jialal’s view.

However, not all data are consistent with the predictive value of CRP. At the 2010 meeting of the American Heart Association, data from the Anglo-Scandinavian Cardiac Outcomes Trial, also a statin treatment trial, found that including screening for hsCRP did not improve conventional heart disease risk assessment in patients with traditional risk factors. The investigators called this finding “surprising” in light of the results of JUPITER.

In a press release the AHA issued on Nov. 17, 2010, lead investigator Peter S. Sever, MD, of Imperial College London, said, “The message coming out of the JUPITER study was that we should be screening people for CRP irrespective of their other risk factors. That’s very, very expensive and almost certainly not a cost-effective intervention, particularly given these findings that measurement of CRP doesn’t add anything in a much more widely representative population” (http://sciencenews.myamericanheart.org/sessions/late_breaking.shtml).

Dr. Jialal says, “These data have not yet been published in a peer-reviewed journal, so we have to wait to comment, and unlike JUPITER, which was a prospective study, this is a retrospective analysis.”

Thus, CRP remains controversial despite compelling data. “The controversy,” says Dr. Wu, “revolves around whether it adds extra value to the traditional markers we already use to indicate inflammation.”

Myeloperoxidase (MPO), a pro-oxidant found in leukocytes, is a less proven but promising CVD marker. MPO oxidation makes atherogenic LDL particles. In a 2001 study, MPO levels were measured in several hundred subjects undergoing cardiac catheterization. MPO in both blood and leukocytes was elevated in coronary artery disease patients (Zhang R, et al. JAMA. 2001;286:2136–2142). In another study, initial baseline MPO level predicted risk for MI in subjects presenting with chest pain, even those who were troponin T-negative (Brennan ML, et al. N Engl J Med. 2003;349:1595–1604). These data have been replicated (e.g.: Baldus S, et al. Circulation. 2003;108:1440–1445).

“In this talk I was teaching pathologists about the future,” Dr. Jialal told CAP TODAY. CRP is the present. It is a standardized assay for which there is a CAP Survey. In contrast, he says, “Myeloperoxidase falls flat on its face. It is not standardized.” There are enzyme assays measuring activity and mass assays. Dr. Jialal calls MPO “a test in evolution.” Its only value now, in his view, is to predict worse mortality in MI patients. “In healthy people myeloperoxidase is not a cost-effective test. If it is validated and standardized, it has great promise in acute coronary syndromes.”

Dr. Wu says both CRP and myeloperoxidase are important in predicting CVD. His take: “MPO is more proximal to the point of plaque rupture and could act as a marker of plaque stability, less so than as a marker of atherosclerosis.” MPO infiltrates plaque and makes it more vulnerable. “Plaque formation and plaque destabilization are two separate steps,” he says. “Data looking at MPO as an early risk-assessment marker are equivocal. It is not there today.”

All of the health problems of CVD are magnified in the metabolic syndrome, lending urgency to the search for markers. “There are no new additional tests for metabolic syndrome,” Dr. Jialal says. “We know how to treat high LDL.” But those with metabolic syndrome might have low HDL-C with normal LDL-C but be at risk because of high triglycerides, high glucose, and central obesity. Metabolic syndrome increases the risk for coronary heart disease twofold and for type 2 diabetes fivefold. “This cluster we don’t know what to do about,” Dr. Jialal says. And this cluster is epidemic: 35 percent of American adults have it. “It’s scary,” Dr. Jialal says.

According to one definition, a person has metabolic syndrome if he or she has three of the five cardinal features: high triglycerides, low HDL-C, high blood pressure, central adiposity, and insulin resistance. Metabolic syndrome is also a proinflammatory state, with CRP and adiponectin implicated.

Like IL-10, adiponectin is an antiinflammatory substance. “Any therapy that raises adiponectin improves insulin resistance,” Dr. Jialal says. Some diabetes drugs in the glitazone class do this. So do diet and exercise. Standardizing assays for adiponectin will take a long time, Dr. Jialal says, partly because it comes in several forms—mono­mers, multimers, and hexamers. He thinks that assays reporting the ratio of high-molecular-weight adiponectin to total adi­po­nectin hold promise but need standardization. His discussion of this problem was published in a 2009 editorial in the American Journal of Clinical Pathology (2009;132: 161–162).

Dr. Jialal also advocates more frequent measurement of protein in urine, for coronary heart disease and diabetes. “We are now learning that in addition to predicting the progression of diabetic nephropathy, microalbuminuria greatly increases the risk of cardiovascular events in nondiabetics also,” Dr. Jialal says. “Looking for protein in the urine is a simple screening test.” Microalbuminuria is defined as 20–200 µg/min on a spot urine specimen or 30–300 µg/day on a 24-hour urine specimen or 30–300 µg/mg creatinine on a timed collection. Spot urine screens can be done on people with hypertension, diabetes, and metabolic syndrome. “It can better inform clinicians about pharmacotherapy,” he says.

Dr. Wu says microalbuminuria assessment has traditionally been used to predict early-stage diabetic nephropathy. “It is certainly linked to the metabolic syndrome, but more to the specific complication of diabetic or prediabetic nephropathy, as enlargement of the glomerular basement membrane leads to leakage of albumin into the urine.” Of course, these features are all linked, Dr. Wu acknowledges. “Whether that is being used as a screening test is something I’m not aware of,” he says. “Certainly it is used for diabetes, but using it for metabolic syndrome is a different concept.”

As Dr. Jialal said, he was exposing pathologists and others in the audience to the possible future of CVD risk assessment, as was Dr. Kroll. It may be that use of microalbuminuria in the metabolic syndrome, or apoB, MPO, or non-HDL-C for CHD prediction, will become standard. Ongoing work with these mechanistically important molecules bears watching.