American Association for Clinical Chemistry
Better health through laboratory medicine
September 2008 Clinical Laboratory News: Apolipoprotein B

 

September 2008: Volume 34, Number 9 


Apolipoprotein B
Is it Time to Switch from LDL-C?

By John H. Contois, PhD, and Joseph P. McConnell, PhD

 

For nearly two decades, LDL-C has been the cornerstone for assessment of cardiovascular disease risk, as well as the primary guide for preventive therapy. But recently, there has been much debate on the merits of monitoring LDL-C versus apolipoprotein B (apo B). Numerous prospective studies have demonstrated that apo B is more strongly predictive of coronary heart disease (CHD) risk than LDL-C (1). In fact, a recent consensus conference report from the American Diabetes Association and the American College of Cardiology recognizes the importance of apo B measurement (2).

In light of the mounting evidence, laboratorians should become more familiar with the clinical data on apo B as a marker of CHD risk. Here, we review the studies of apo B reported to date and discuss potential advantages of apo B measurement over LDL-C.

Problems with LDL-C

Cholesterol serves as a useful surrogate for estimating LDL particles, but LDL-C concentration can vary widely between individuals with the same LDL particle concentration. In one study, researchers found that the ratio of cholesterol to triglycerides in LDL particles can vary from 1.8 to 11.5 between individuals (3). The majority of subjects, 118 healthy men and women, had large LDL particles with the expected ratio of cholesterol to triglyceride >4. But surprisingly, 21% of subjects had LDL particles that were cholesterol-depleted (cholesterol/triglyceride ratio < 4), indicating that even an accurately measured LDL-C will underestimate LDL particle concentration and, presumably, CHD risk. LDL-C content does not reflect LDL particle concentration because intravascular metabolism can alter both lipoprotein size and lipid composition.

LDL-C Analytical Issues

Although LDL-C measurement is the gold standard for assessing CHD risk, the calculations and assays are not without flaws. Traditionally, LDL has been defined as the lipoprotein fraction in the density range from 1.019 to 1.063 g/mL determined by sequential density ultracentrifugation. Lipoprotein (a) (Lp(a)), with a density range of 1.045 to 1.080 g/mL, overlaps with LDL. Later, the beta-quantification method defined LDL-C as the cholesterol in the density fraction >1.006 g/mL minus the cholesterol in the HDL fraction isolated by precipitation. This method, therefore, actually measures intermediate density lipoprotein (IDL) and lipoprotein(a) (Lp(a)) cholesterol along with LDL-C.

The Friedewald equation, which estimates LDL-C, also includes the IDL and Lp(a)-C components and assumes a standard very low density lipoprotein (VLDL) triglyceride/cholesterol ratio, a lack of chylomicrons, and a lack of excessive remnant lipoproteins. Therefore, it cannot be used if patients are nonfasting, when triglycerides are > 400 mg/dL, or if the patient has type III hyperlipoproteinemia. The accuracy of the Friedewald equation begins to fail when triglycerides are >200 mg/dL and the effect is more pronounced at lower LDL-C concentrations. Furthermore, the equation is based on the measurement of total cholesterol, triglycerides, and HDL-C, and therefore is affected by inaccuracies associated with each of the three methods.

There is also a high degree of variation among manufacturers’ direct LDL-C assays. Although these assay methods are largely unaffected by fasting status, the exisiting variability highlights the need for better standardization. Currently, LDL-C assays are not standardized to a common reference material, but instead rely upon comparison to a reference method. The variability with LDL-C assays, however, appear to relate more to inherent assay design than to calibration.

LDL-related CVD Risk

LDL particles, not simply LDL-C, play a central role in atherogenesis. The initiating process is the subendothelial retention of intact apo B-containing particles (4). LDL particles move into the arterial intima through a gradient-driven process, and the rate of passive diffusion is increased when the concentration of circulating LDL particles is elevated. Once inside the intima, the LDL particles bind to proteoglycans and initiate a process whereby the LDL particles are oxidized or otherwise modified and are taken up by monocytes to form macrophages. The cholesterol molecules contained in the LDL particles are passengers, but the intact particles drive the process.

Although apo B is widely considered a distinct risk factor for CHD, it is simply an alternate measure of LDL-related risk because it largely reflects LDL particle concentration. LDL-C, non-HDL-C, and total apo B are, to varying degrees, all measures of LDL-related cardiovascular risk. They are all highly interrelated, and so they have all been implicated as predictors of CHD in epidemiological studies. But biologically, they reflect different metabolic processes. Despite a high correlation with CHD risk, these markers are only modestly concordant, indicating that one cannot substitute for another in classifying patients into risk categories. At any given LDL-C level, apo B concentration can vary widely, and vice-versa.

An Overview of ApoB

The apoB gene, located on the short arm of chromosome 2, is 43 kb long with 29 exons. The protein itself is made up of 4,536 amino acids with a molecular weight of about 550 kDa. The two sites of apo B synthesis are the liver, which creates apo B-100, and the intestine, which produces apoB-48. A C-to-T substitution at nucleotide 6666 inserts a stop codon into the apo B mRNA and is responsible for the apoB-48 protein of 265 kDa.

Apo B is a component of all atherogenic or potentially atherogenic particles, including VLDL-, IDL, LDL, and Lp(a). Each of these particles contains one molecule of apo B, making it a direct measure of the number of atherogenic lipoprotein particles in the circulation. However, even in hypertriglyceridemic patients, the vast majority of total plasma apo B is associated with LDL, making apo B an effective surrogate for LDL particle concentration.

The Adult Treatment Panel of the National Cholesterol Education Program (NCEP) suggests an LDL-C goal of <100 mg/dL and a non-HDL-C goal of <130 mg/dL in high-risk patients. An equivalent goal for apo B, <90 mg/dL, has been proposed (5). Stein and colleagues have assessed the comparability of these goals using a database of more than 22,000 individuals from clinical trials. In 14,425 subjects with normal triglycerides (<200 mg/dL), 58% and 66% met the LDL goal and the non-HDL cholesterol goal, respectively. However, only 30% of these same individuals met the apo B goal. In 7,611 subjects with elevated triglycerides, only 17% met the apo B goal, while 60% and 51% met the LDL-C and non-HDL-C goals, respectively. Interestingly, the subjects who met the apo B goal were virtually assured of meeting both the LDL and non-HDL goals (6).

In contrast to LDL-C, standardization of apo B assays has made much progress. An IFCC standards committee recognized that bias between manufacturers for apo B was due to a lack of common calibration material. The committee identified a suitable reference material that manufacturers can use to assign values to calibration materials. Subsequent studies have reported a very respectable inter-laboratory CV of 3.1%–6.7% among assays from manufacturers using fresh-frozen patient sera and common calibrators (7).

Furthermore, fasting is not required for apo B measurement. Despite laboratorians’ frequent objection that availability of apo B assays is limited, immunoturbidimetric assays have become more widely available for use on a variety of automated platforms.

A Look at Lipoprotein Disorders

Many lipoprotein disorders are characterized by elevated serum apo B concentration. Apo B mediates the uptake of LDL by liver and peripheral tissue via a specific interaction with the LDL receptor. Familial hypercholesterolemia (FH) is due to a defect in the LDL receptor that prevents the clearance of LDL particles from the circulation. An increased number of LDL particles is therefore a hallmark of FH.

Familial defective apo B is a related disorder resulting from a mutation in apo B that prevents binding of the protein to the LDL receptor, resulting in a clinical phenotype similar to FH. Sporadic or polygenic hypercholesterolemia is likely due to overproduction of LDL particles. Hypertriglyceridemia (HTG) with elevated LDL particle concentration, and therefore higher apo B levels, may be the most common dyslipidemia. However, HTG without an elevated LDL particle concentration is probably not atherogenic. Similarly, individuals with Lp(a) excess also appear to have an excess of small, dense LDL particles (8) .

The most common and perhaps underdiagnosed lipoprotein disorder, familial combined hyperlipoproteinemia (FCH), was originally defined as a total cholesterol or triglyceride concentration ≥95th percentile in probands with premature CHD and at least one first-degree relative. Subsequent research has identified an association of FCH with an increase in small, dense LDL particles and demonstrated that FCH is most accurately diagnosed with a panel that includes measurement of apo B (9).

Prospective Studies of Apo B in Primary and Secondary Prevention

In a meta-analysis of apo B prospective studies, Thompson and Danesh found that apo B is a significant predictor of CHD with an overall relative risk of about 2.0 for the upper versus the lower tertile (10).

Another compelling study that supports use of apo B is the AMORIS study, which followed more than 175,000 men and women over the age of 60 for 5 years, including 864 men and 359 women who suffered a fatal MI (11). After adjustment for age and traditional lipid risk factors, including LDL-C, apo B remained a significant predictor of fatal MI, with relative risks of 1.33 (CI 1.17–1.51) and 1.53 (CI 1.25–1.88) for an increase of one standard deviation in men and women, respectively. LDL-C was an insignificant risk factor in women and only modestly associated with MI in men.

In the Quebec Cardiovascular Study of 2,039 men, ages 45–76, apo B was a strong, independent predictor of future cardiac events even after adjustment for age, smoking, systolic blood pressure, diabetes, and medication use (12). Interestingly, the investigators found a synergistic relationship between apo B and the total cholesterol/HDL-C ratio (TC/HDL). When the TC/HDL ratio was low, an elevated apo B was associated with a 60% increased risk of CHD. But when the TC/HDL ratio was high, an elevated apo B was associated with a 2.6-fold increased risk. A 13-year follow-up of the participants also suggested a similar synergy between LDL-C and apoB (13). Among the men with elevated LDL-C but low apo B levels (<128 mg/dL), relative risk for CHD was a modest 1.5. But when both LDL-C and apo B were elevated, the relative risk was 2.2.

Among the published prospective studies of apo B in primary prevention, all but one found a statistically significant association with CHD, even after adjustment for nonlipid risk factors. Of the 13 primary prevention studies that also provided data for LDL-C, only nine reported a significant relationship between LDL-C and CHD in both men and women or all subjects combined. Among the studies reporting both apo B and LDL-C, apo B was consistently the stronger risk factor.

The secondary prevention studies reveal similar results. Baseline value of apo B was a significant predictor of recurrent cardiovascular events in all but one study, including the 4S, LIPID, THROMBO, and other studies. Neither apo B nor LDL-C was a significant predictor of recurrent events in the VAHIT study; however, subjects were selected to have relatively low LDL-C concentrations.

There is a wide variation in the reported relative risks for CHD in these epidemiologic studies, largely dependent on whether apo B levels are adjusted for other lipids and lipoproteins. Consequently, the debate about the utility of apo B for CHD risk assessment has become one of statistics rather than biological plausibility. However, as the Quebec Cardiovascular Study and AMORIS have shown, in large-scale studies with precise and standardized apo B measurement, apo B does appear to have statistical significance even when traditional lipids and lipoproteins are covariates in the regression models. This is also evident in the Health Professionals Follow-up Study. When apo B and LDL-C were simultaneously included in the model, relative risk was strongly associated with apo B, while LDL-C and non-HDL-C were no longer statistically significant (14).

Monitoring LDL-lowering Therapy

Statins have proven to be highly effective in reducing serum cholesterol through inhibition of HMG-CoA reductase. Clinical trials have consistently shown that a remarkable lowering of LDL-C is associated with a substantial lowering of relative CHD risk; however, in terms of absolute risk, reduction is far less dramatic. This has led many lipid experts to conclude that LDL-C targets need to be set much lower.

However, it appears that a reduction in apo B or LDL particles may be a better target for monitoring therapeutic effectiveness and residual risk. Statins also reduce the production of both VLDL-apoB and LDL-apo B. But as shown in Table 1, the reduction in serum apo B concentration is not as dramatic as the LDL-C decrease, and levels of apo B in patients on statin therapy indicate potential residual risk associated with increased LDL particles (15).

Table 1
Effect of Statins on LDL-C, Non-HDL-C, and Apo B

 

LDL-C

Non-HDL-C

Apo B

 

Baseline (mmol/L)

Reduction at 54 weeks (%)

Baseline (mmol/L)

Reduction at 54 weeks (%)

Baseline (mmol/L)

Reduction at
54 weeks (%)

Atorvastatin

4.60

42.1

5.58

38.4

4.39

31.9

Fluvastatin

4.63

29.0

5.61

25.7

4.34

18.9

Lovastatin

4.60

35.5

5.61

31.8

4.39

25.4

Pravastatin

4.63

28.1

5.61

25.5

4.34

18.7

Simvastatin

4.55

25.6

5.50

32.0

4.29

25.0

From: Walldius and Jungner, J Intern Med 2004; 255:188–205.

In the Air Force/Texas Coronary Atherosclerosis Prevention Study (AFCAPS/TexCAPs) apo B at baseline and following 1 year on therapy was a strong predictor of future CVD events, whereas LDL-C failed to reach significance (p > 0.05 at baseline and on therapy) (16). A similar analysis in the Long-term Intervention with Pravastatin in Ischemic Disease (LIPID) study showed similar findings (17). The reason is apparent: LDL-related risk is not captured by LDL-C measurement alone. Results from both primary and secondary statin trials suggest that on-therapy concentrations of apo B or LDL particles better predict future CHD events than does LDL-C.

New Directions

Numerous prospective epidemiologic studies show that apo B is a statistically significant predictor of future fatal and nonfatal heart disease. Apo B measurement to assess CHD risk is especially important in the large and rapidly growing subset of the population with characteristics of the metabolic syndrome and in diabetic patients. Individuals with the metabolic syndrome or diabetes tend to have an increased number of small, dense LDL particles but relatively normal LDL-C levels. Furthermore, therapy with HMG-CoA reductase inhibitors reduces LDL-C to a greater extent than LDL particle concentration, suggesting that apo B may provide a better assessment of residual risk for patients on this therapy.

Clearly, for apo B to become an important complement to the standard lipid profile, the medical community will need to accept that the number of circulating LDL particles is the major component of LDL-related CHD risk, not LDL-C. The movement towards this has already started. In a recent review, a panel of experts concluded that CHD risk is more directly related to the number of circulating atherogenic particles than to the cholesterol content of lipoproteins, and the panel advocated apo B measurement (1). Furthermore, a consensus conference report from the American Diabetes Association and the American College of Cardiology concluded that measurement of apo B with a standardized assay is warranted in patients with metabolic syndrome, especially to assist with therapeutic monitoring (2). Table 2 gives the suggested treatment goals for these patients.

Table 2
Suggested Treatment Goals in Patients with Cardio-metabolic Risk and Lipoprotein Abnormalities

 

Goals (mg/dL)

 

LDL
Cholesterol

Non-HDL
Cholesterol

Apo B

Highest-risk patients, including those with (1) known CVD or (2) diabetes plus one or more additional major CVD risk factor*

<70

<100

<80

High-risk patients, including those with (1) no diabetes or known clinical CVD but two or more additional major CVD risk factors or (2) diabetes but not other major CVD risk factors

<100

<130

<90

*Other major risk factors (beyond dyslipoproteinemia) include smoking, hypertension, and family history of premature CAD.

From: Brunzell et al., JACC 2008;51:1512.

Canadian medical practice has already added apo B to guidelines for assessment of at risk patients (18), and updated guidelines from the National Cholesterol Education Program are expected in 2009 on cholesterol testing and management. The addition of apo B to these guidelines seems to be the logical next step. As laboratorians, our community will need to stay abreast of these developments, which are likely to create major changes in patient management.

REFERENCES

  1. Barter PJ, Ballantyne CM, Carmena R, Castro Cabezas M, Chapman MJ, et al. Apo B versus cholesterol in estimating cardiovascular risk and in guiding therapy: report of the thirty person/ten-country panel. J Intern Med 2006;259:247–258.
  2. Brunzell JD, Davidson M, Furberg CD, Goldberg RB, Howard BV, Stein JH, Witztum JL. Lipoprotein management in patients with cardiometabolic risk: conference report from the American Diabetes Association and the American College of Cardiology Foundation. JACC 2008;51:1512–1524.
  3. Otvos JD. Measurement of triglyceride-rich lipoproteins by nuclear magnetic resonance spectroscopy. Clin Cardiol 1999;22(6 Suppl):II21–27.
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  5. Grundy SM. Low density lipoprotein, non-high density lipoprotein, and apolipoprotein B as targets for lipid-lowering therapy. Circulation 2002;106:2526–2529.
  6. Stein EA, Sniderman A, Laskarzewski P. Assessment of reaching goal in patients with combined hyperlipidemias: low density lipoprotein cholesterol, non-high-density lipoprotein cholesterol, or apolipoprotein B. Am J Cardiol 2005;96[suppl]:36K–43K.
  7. Marcovina S, Packard CJ. Measurement and meaning of apolipoprotein B plasma levels. J Intern Med 2006;259:437–446.
  8. Zambon A, Braun BG, Deeb SS, Brunzell JD. Genetics of apolipoprotein B and apolipoprotein AI and premature coronary artery disease. J Intern Med 2006;259:473–480.
  9. Veerkamp MJ, de Graaf J, Hendriks JCM, Demacker PNM, Stalenhoef AFH. Nomogram to diagnose familial combined hyperlipidemia on the basis of results of a 5-year follow-up study. Circulation 2004;109:2980–2985.
  10. Thompson A, Danesh J. Association between apolipoprotein B, apolipoprotein AI, the apolipoprotein B/AI ratio and coronary heart disease: a literature-based meta-analysis of prospective studies. J Intern Med 2006;259:481–492.
  11. Walldius G, Jungner I, Holme I, Aastveit AH, Kolar W, Steiner E. High apolipoprotein B, low apolipoprotein A-1, and improvement in the prediction of fatal myocardial infarction (AMORIS study): a prospective study. Lancet 2001;358:2026–2033.
  12. Lamarche B, Moorjani S, Lupien PJ et al. Apoprotein A-1 and B levels and the risk of ischemic heart disease during a 5 year follow-up of men in the Quebec Cardiovascular Study. Circulation 1996;94:273–278.
  13. St-Pierre A, Cantin B, Dagenais GR et al. Low-density lipoprotein subfractions and the long-term risk of ischemic heart disease in men. 13-year follow-up data from the Quebec Cardiovascular Study. Arterioscler Thromb Vasc Biol 2005;25:553–559.
  14. Pischon T, Girman CJ, Sacks FM, Rifai N, Stampfer MJ, Rimm EB. Non-high density lipoprotein cholesterol and apolipoprotein B in the prediction of coronary heart disease in men. Circulation 2005;112:3375–3383.
  15. Walldius G, Jungner I. Apoplipoprotein B and apolipoprotein AI: risk indicators of coronary heart disease and targets for lipid-modifying therapy. J Intern Med 2004;255:188–205.
  16. Gotto AM, Whitney E, Stein EA, Shapiro DR, Clearfield M, Weis S. Relation between baseline and on-treatment lipid parameters and first acute major coronary events in the Air force/Texas Coronary Atherosclerosis Prevention Study (AF-CAPS/TexCAPS). Circulation 2000;101:477–484.
  17. Simes RJ, Marschner IC, Hunt D, Colquhoun D, Sullivan D, Stewart RAH. Relationship between lipid levels and clinical outcomes in the long-term intervention with pravastatin in the ischemic disease (LIPID) trial. To what extent is the reduction in coronary events with pravastatin explained by on-study lipid levels? Circulation 2002;105:1162–1169.
  18. Genest J, Frolich J, Fodor G, McPherson R, the Working Group on Hypercholesterolemia and Other Dyslipidemias. Recommendations for the management of dyslipidemias and the prevention of cardiovascular disease: summary of the 2003 update. JAMC 2003;169:921–924.

John H. Contois, PhD, DABCC, FACB is Manager of Research and Development at Maine Standards Company LLC, Windham, Maine.

 

 


 

Joseph P. McConnell, PhD, DABCC, is the Laboratory Director of Cardiovascular Laboratory Medicine, in the Department of Laboratory Medicine and Pathology at the Mayo Clinic, Rochester, Minn.