Nonlipid Serum Markers for Clinical Risk Assessment of Coronary Artery Disease

Prevention has become an important component in the management of cardiovascular disease, a leading cause of death in the United States. Success with modification of traditional risk factors?hypertension, diabetes, dyslipidemia, smoking, and obesity?has led researchers to investigate additional factors that may identify people at risk for a coronary event. C-reactive protein, the most studied and valuable nonlipid serum marker identified thus far, has shown to predict risk for cardiovascular events. Other risk factors studied include homocysteine, fibrinogen, and low-density lipoprotein oxidation; these have shown some clinical benefit in the early phases of research. Interventions that modify these markers, including statin therapy, may need to be considered. Exciting research is ongoing that may crystallize the role of these novel factors in the management of coronary artery disease.

Hetal A. Gandhi, MD,

Resident; Department of Internal Medicine; Mayank K. Shah, MD, Clinical Instructor, Department of Family Medicine, Lutheran General Hospital, Park Ridge, Ill

Risk factor modification has become a cornerstone of the management of coronary artery disease (CAD). Perhaps this explains part of the significant reduction that has occurred in CAD mortality since 1970. However, CAD remains a significant burden to society, accounting for more than 1.4 million deaths every year.¹ As a result, aside from the established risk factors of hypertension, smoking, diabetes, dyslipidemia, obesity, and family history of premature CAD, interest is growing in identifying nonlipid markers that may predict risk for cardiac events. Researchers have identified more than 100 such markers; only a few have thus far been demonstrated to have clinical implications?among them are C-reactive protein (CRP), homocysteine, fibrinogen, and oxidation of low-density lipoprotein (LDL). Physicians must be aware of these new risk factors and their role in the development of CAD, because one fourth of patients who have a coronary event have minimal or none of the established risk factors.² The relative contribution of novel risk factors is likely to increase over the next few years as research continues.

C-Reactive Protein

The acute-phase response is a major pathophysiologic phenomenon that accompanies acute or chronic inflammation. Acute-phase proteins are defined as proteins whose plasma concentrations increase or decrease by at least 25% during inflammatory states. Despite the lack of diagnostic specificity, measurement of serum levels of acute-phase proteins is useful, because it may reflect the presence and intensity of an inflammatory process. The most extensively studied indicator of the acute-phase response in cardiovascular disease (CVD) is CRP. A standardized, high-sensitivity CRP (hs-CRP) assay is now widely available to measure CRP serum levels. The presence of inflammation has been noted since the earliest histologic observations and theories on the development of atherosclerosis. An acute-phase reactant, hs- CRP plays an important role in the innate immune response and is now recognized as a mediator of atherothrombotic disease.³ Recent evidence has shown that CRP directly participates in the atherothrombotic process by activating complement, regulating endothelial nitric oxide (NO) synthase expression and NO synthesis, upregulating expression of cellular adhesion molecules, and possibly directly modulating oxidation of LDL (Figure 1). ³

To date, more than a dozen prospective epidemiological studies of individuals with no history of CVD have demonstrated that a single, nonfasting measurement of CRP predicts the incidence of CAD,⁴ stroke,⁵ peripheral artery disease,⁶ and sudden cardiac death.⁷ It applies to both women and men, the elderly and middle aged, smokers and nonsmokers, and persons with and without diabetes.⁴ CRP levels have also been shown to predict risk of recurrent ischemia and death in patients with stable and unstable angina or with acute coronary syndromes, and those who are undergoing percutaneous angioplasty or coronary artery bypass grafting.⁴

Current data suggest that patients with negative troponin and CRP levels in the emergency department are unlikely to have flow-limiting CAD.⁸ An analysis of women with the metabolic syndrome showed that those with the highest CRP levels (ie, >3 mg/L) were 2.1 times more likely to have a cardiovascular (CV) event (ie, myocardial infarction [MI], stroke, coronary revascularization, CV death) than those with the lowest levels (ie, <1 mg/L).⁹ The Women's Health Study, with 27,939 participants, showed that CRP level was a better predictor of CV events than LDL cholesterol (LDL-C) level, and it adds prognostic information at all levels of calculated Framingham risk.¹⁰ At 8 years, women in the lowest quintile of serum CRP(ie, ?0.5 mg/L) had a 0.9% relative risk of a first CV event, whereas those in the lowest quintile of LDL-C (ie, ?97.6 mg/L) had a 1.4% relative risk.¹⁰

CRP measurements

The measurement of CRP has been standardized. The American Heart Association (AHA) and the Centers for Disease Control and Prevention indicate that results of hs-CRP testing should be expressed as milligram per liter (mg/L), with the following definitions signaling risk for CVD:

• Average risk, CRP 1-3 mg/L
• If CRP >10 mg/L, repeat the test and consider additional evaluation to determine if infection or inflammation is the cause.¹¹

Some issues affect the utility of hs-CRP measurement, however. First, there is lack of specificity for atherosclerotic disease.¹¹ CRP is an acute-phase reactant, and levels rise with all inflammatory disorders. Thus, elevated values can be considered predictive of CV risk only if there is no apparent cause.¹¹ Second, there is a lack of well-established evidence for measurement of hs-CRP in adults at lower CV risk (Framingham risk, <10% in 10 years).¹¹ Finally, there is as yet no evidence that treatment strategies based on CRP level improve survival.¹¹ However, emerging data may support routine use of hs-CRP level in risk assessment and treatment selection in secondary prevention.

Despite its limitations, CRP measurement is useful in specific situations. For example, in a patient at intermediate risk for CAD (calculated 10-year Framingham risk, 10%-20%), the hs-CRP test may help direct further evaluation and therapy for primary prevention.¹¹ There is insufficient evidence to suggest that hs-CRP may be used to track the efficacy of treatment,¹¹ or to support the use of CRP for primary prevention, but the evidence may eventually extend to primary prevention.

Intervention to reduce CRP levels

Many interventions known to reduce CV risk have been linked to lower CRP levels. In particular, weight loss; low-fat, low-calorie diet; exercise; and smoking cessation all reduce levels of CRP and CV risk.⁴ Randomized clinical trials involving statin therapy have shown that, on average, median CRP levels decline 15% to 25% as early as 6 weeks after initiation of therapy.⁴ In the Air Force/Texas Coronary Atherosclerosis Prevention Study, lovastatin (Mevacor) was very effective in those with elevated CRP levels, even when LDL-C levels were below the thresholds set by the National Cholesterol Education Program Adult Treatment Panel III guidelines.¹²Arecent study of more than 3700 patients with established CAD showed that reduction in CRP levels using statin therapy resulted in decreased risk of recurrent MI or death from coronary causes, independent of reduction in LDL.¹³

Many other drugs?such as aspirin, clopidogrel (Plavix), and the thiazolidinediones?have also been shown to reduce CRP levels.⁴

Homocysteine

Homocysteine, an intermediate compound derived from methionine, is metabolized via 2 pathways: vitamin B6-dependent transsulfuration and vitamin B₁₂-and folate-dependent remethylation (Figure 2).¹⁴ Elevations in homocysteine can occur for several reasons (Table).¹⁵ Evidence shows that homocysteine has primary atherogenic and prothrombogenic properties.¹⁶ Several effects may contribute to its role in vascular disease. Hyperhomocysteinemia is associated with endothelial dysfunction and endothelial cell injury, enhanced thromboxane A₂ formation and platelet aggregation, proliferation of vascular smooth muscle cells, and procoagulant activity.

MTHFR

MTHFR

MTHFR

Most cross-sectional and retrospective studies have shown an association between plasma homocysteine levels and CVD.¹⁵ A meta-analysis of 27 observational studies involving approximately 4000 patients showed that hyperhomocysteinemia increased risk of fatal and nonfatal CAD, cerebrovascular disease, and peripheral vascular disease.¹⁷ A meta-analysis of data from 40 observational studies involving 11,162 patients with the 677C?T genotype (a genetic alteration in an enzyme involved in folate metabolism that causes elevated homocysteine levels) and 12,758 matched controls demonstrated that individuals with the 677 TT genotype had a 16% higher risk of CAD than those with the 677 CC genotype, particularly in individuals with low folate levels.¹⁸

The results of prospective studies have not been uniform. Although a significant association between elevated homocysteine levels and CVD has been reported,^19,20 some large prospective studies have failed to show this connection.^21,22 An analysis of data of nearly 15,000 physicians from the Physicians' Health Study (PHS) failed to demonstrate a significant association between plasma homocysteine levels and risk of MI or CV death.²³

MTHFR

Normal fasting plasma homocysteine levels usually range from 5 to 15 ?mol/L. Hyperhomocysteinemia may be classified as moderate (16-30 ?mol/L), intermediate (31-100 ?mol/L), or severe (>100 ?mol/L).¹⁴ There are several arguments against screening the general population. First, for individuals with the TT genotype of 677, the population-attributable risk is only 1% to 2%, and adequate folic acid intake further reduces the impact on CV outcomes, including fatal and nonfatal CAD, cerebrovascular disease, and peripheral vascular disease. Therefore, provision of adequate folic acid is probably more important than additional homocysteine measurements or genetic testing for such persons.¹⁵ It remains to be demonstrated that lowering plasma homocysteine levels with diet or vitamin therapy will reduce CV mortality and morbidity.¹⁴ However, even if treatment of hyperhomocysteinemia is beneficial, it may be more cost-effective to recommend daily multivitamin and folic acid supplementation.¹⁴

Ongoing clinical trials are evaluating the effects of folic acid and vitamins B6 and B₁₂ on CVD. In the absence of stronger evidence, routine measurement of homocysteine levels is currently not recommended for CV risk assessment; instead, physicians should focus on meeting patients' current recommended dietary allowances for folate and vitamins B6 and B₁₂ through food, including vegetables, fruits, meats, fish, and fortified grains and cereals.¹⁴

Homocysteine measurements may be important when traditional risk factors do not appear to account for an increased vascular risk.¹⁴ An AHA advisory panel also recommends screening for serum homocysteine level in patients with malabsorption syndromes, malnutrition, hypothyroidism, renal failure, or systemic lupus erythematosus and in those taking medications, such as nicotinic acid, theophylline (eg, Bronkodyl, Elixophyllin), methotrexate (Rheumatrex, Trexall), bile acid resins, or levodopa (Larodopa).¹⁴ In such patients, it may be advisable to increase the intake of vitamin-fortified foods or to supplement with daily folic acid and vitamins B6 and B₁₂.¹⁴

Fibrinogen

Thrombogenic factors are essential in the pathogenesis of coronary atherosclerosis.²⁴ Elevated plasma fibrinogen, one of the chief components of the coagulation system of the body, has been identified in several studies as a predictor of CVD.^25,26 Fibrinogen is a plasma protein synthesized in the liver. In addition, fibrinogen plays an important part in platelet aggregation, increased blood viscosity, fibrin formation, and may have mitogenic and angiogenic properties.^24,27 It is also an acute-phase reactant whose levels are increased in inflammatory states.²⁷

Many prospective studies have identified an association between plasma fibrinogen concentration and CVD. In another analysis of data from the PHS, those with high fibrinogen levels (after adjusting for LDL and total cholesterol, other coronary risk factors, and aspirin therapy) had a 2-fold increase in MI risk.²⁵ Data from the Framingham Offspring Population study of 2632 adults suggest a strong relationship between fibrinogen levels and traditional CV risk factors, including age, smoking, diabetes, total cholesterol, high-density lipoprotein cholesterol (HDL-C), and triglycerides.²⁶ Fibrinogen levels were higher in those with CVD than in those without. These data suggest that elevated fibrinogen concentration may be a mechanism by which traditional risk factors exert their effects to contribute to CV events.

High fibrinogen levels are associated with increased age, high LDL-C and triglyceride concentrations, low HDL-C concentration, smoking, diabetes, stress, obesity, family history of premature CAD, physical inactivity, and socioeconomic factors (eg, low education, poverty).¹⁶ Smoking cessation, weight loss, regular exercise, and moderate alcohol consumption are known to reduce plasma fibrinogen values. Because of a high correlation between fibrinogen and major CVD risk factors, measures recommended to control CAD will also reduce elevated fibrinogen levels.²⁸

The clinical benefit of fibrinogen measurements is uncertain. A single fibrinogen reading may not be adequate for the prediction of CV risk. One study evaluated the accuracy of triplicate measurements of fibrinogen at baseline and at 3 months on CV risk in 60 healthy adults.²⁹ However, only 55% were appropriately assigned to the accurate fibrinogen tertile (low, moderate, or high). Fibrinogen measurements also varied by more than 10% in 45% of subjects and by 5% in 80% of subjects.²⁹ Intraindividual variability in fibrinogen measurements (even when 3 measurements taken from replicate samples are averaged), the lack of a single standardized assay, and the lack of conclusive evidence to show that reducing fibrinogen levels improves CV risk have limited the use of fibrinogen measurements as a clinical screening tool.^29,30

LDL Oxidation

Agents in vascular tissue and inflammatory cells are responsible for the oxidative modification of LDL, which is thought to play an important part in the pathogenesis of atherosclerosis.³¹ The susceptibility of LDL to oxidation is evaluated by studying the kinetics of in vitro oxidation products.³⁰ Oxidized LDL acts as a chemoattractant for T lymphocytes and monocytes, immobilizes macrophages within the cell wall, increases LDL uptake by the macrophages, promotes the formation of foam cells, and has a direct cytotoxic effect on subendothelial and smooth muscle cells (Figure 3).³¹ No studies have established a role for oxidized LDL in predicting CV risk. There is little agreement on which variable best measures LDL oxidizability or whether any in vitro measure accurately estimates in vivo LDL oxidizability or predicts clinical outcome.³⁰

The oxidative-modification hypothesis of atherosclerosis (ie, oxidative modification of LDL as the initiator of atherosclerosis) has prompted the study of antioxidant vitamins in the prevention, initiation, and progression of CVD. It had been thought that antioxidants capable of inhibiting oxidation of LDL might help reduce the incidence or progression of atherosclerosis.³¹ Several prospective studies have reported an association between the use of vitamin C,³² vitamin E,³³ and beta-carotene³⁴ and lower risk of CVD. However, randomized, controlled trials have failed to demonstrate a consistent role for one or a combination of these vitamins in the prevention of CV events. The US Preventive Services Task Force found insufficient evidence to recommend for or against the use of vitamin A, C, or E supplementations for the prevention of CVD.³⁵ Some studies suggest that antioxidants, such as omega-3 fatty acids or diets rich in oxidation-resistant monounsaturated fat, may have cardioprotective properties.¹⁶

Another emerging area is lipoprotein particles, called apolipoproteins. Lipoprotein(a) is an LDL particle bound to apolipoprotein(a), a protein belonging to the plasminogen family. These particles have been linked to atherosclerosis and thrombosis. Many studies have evaluated the predictive role of lipoprotein( a) for CVD,¹⁶ with conflicting results. Some data suggest that lipoprotein(a), semiquantified by lipoprotein electrophoresis, may have predictive potential for all CV events,¹⁶ but other studies did not confirm these findings.³⁶ No recommendation exists about routine measurement of lipoprotein(a) using lipoprotein electrophoresis.¹

Conclusion

The emergence of novel risk factors has added new enthusiasm to the search for ways to prevent CAD. The success of preventive measures against established risk factors justifies this enthusiasm. Very recent data have shown that CRP has a clinical role as an independent risk factor. Physicians may now be able to modify their patients' CRP levels with lifestyle modifications as well as medications. Despite considerable data associating new risk factors with CVD, no specific recommendations are available. There is also a lack of standardized assays for some risk factors. Finally, the cost-effectiveness of screening and modifying these risk factors must be determined. Despite these limitations, recognizing these novel risk factors may help motivate patients to adopt lifestyle modifications to reduce their risk for CVD.

SELF-ASSESSMENT TEST

1. A single, nonfasting measurement of CRP can predict all the following, except:

• Peripheral artery disease
• Sudden cardiac death

2. All these interventions have been shown to reduce elevated CRP values, except:

• Clopidogrel
• Folate supplementation

3. Which of the following CRP values indicates that a patient is at average risk for CVD?

• 1.0 mg/L
• 4.5 mg/L

4. Which of the following statements about homocysteine is NOT true?

• Prospective studies have consistently shown an association between hyperhomocysteinemia and CVD
• Routine measurement of homocysteine concentration for CV risk assessment is not recommended

5. Increased fibrinogen concentration is associated with all of the following factors, except:

• Vitamin B₁₂ deficiency
• Poverty

(Answers at end of reference list)

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Answers:

1. C; 2. D; 3. B; 4. B; 5. B