**4. ApoB versus non-HDL-C**

ApoB indicates the number of atherogenic particles whereas non-HDL-C indicates the C mass from all atherogenic fractions like VLDL, IDL and the large buoyant LDL and the most atherogenic sdLDL fractions. But is apoB similar to or better than non-HDL-C in predicting risk? Although there is a similarity between apoB and non-HDL-C, they may have different metabolic fate and thus impact on risk. The rationale for using non-HDL-C is based on the fact that there is a close relationship between non-HDL-C and apoB values. Usually the correlation is about 0.80–0.85. However, correlation is not the same as concordance. In fact, two variables can be highly correlated but also be highly discordant, i.e. they do not correspond well. Either they are too high or too low compared with the other variable. Importantly, discordance produces major errors in the middle of the population distribution. Sniderman has frequently presented data with explanations of the advantages of apoB over LDL-C and non-HDL-C (4,13,14,21,62,63). Commonly, the sdLDL-particles contribute much to the large numbers of atherogenic particles, i.e. the apoB number is high and these small particles can easily penetrate into the arterial wall. However, in conditions with high non-HDL-C due to high VLDL-C and high large buoyant LDL-C the sdLDLparticles may be rather low in numbers indicating comparatively low numbers of apoB particles. These larger cholesterol-containing VLDL and IDL particles, although rich in C, do not easily penetrate into the arterial wall.

Of interest, the number of apoB is more closely associated with insulin resistance or markers of the metabolic syndrome than either LDL-C or non-HDL-C (3,62,63). Thus, in patients with hypertriglyceridaemia with normal, or even low LDL-C values, i.e. patients with the metabolic syndrome (MetS), and in patients with overt diabetes, apoB has been shown to be superior to non-HDL-C in predicting vascular risk (3,62,63). Again, even if non-HDL-C and apoB correlate, they are not the same biologically or clinically. In most cases apoB is associated with a higher CV risk than non-HDL-C as well as LDL-C. Furthermore, non-HDL-C may not be that easy to understand or explain for the clinician or the patient once they have learnt that the bad C is LDL-C.

Many studies and clinical trials have been published showing that apoB has a stronger capacity to identify all different phenotypes and to better predict CV risk than both LDL-C and non-HDL-C (3,11,61-63). Sniderman et al. (62) have published a convincing metaanalysis of results from published epidemiological studies that contains estimates of the relative risks of LDL-C, non-HDL-C and apoB of fatal or non-fatal ischemic CV events. Twelve reports including 23,455 subjects and 22,950 events, were analyzed. Whether analyzed individually or in head-to-head comparisons, apoB was the most potent marker of CV risk RR = 1.43; (95% CI, 1.35-1.51), LDL-C was the least RR = 1.25; (1.18-1.33), and non-HDL-C was intermediate RR = 1.34; (1.24-1.44). Only HDL-C accounted for any substantial portion of the variance of the results among the studies. They commented that in patients in whom LDL composition is normal, the cholesterol markers and apoB are equivalent markers of risk, i.e. correlation between the three markers is high. However, when the markers are discordant, that is, when LDL-C is normal but LDL-particles (P)(= apoB) is high or, alternatively, when LDL-C is high but LDL-P are normal, then apoB and non-HDL-C are better markers of risk than LDL-C. They calculated the number of clinical events prevented by a high-risk treatment regimen of all those >70th percentile of the US adult population using each of the 3 markers. Over a 10-year period, a non-HDL-C strategy would prevent 300,000 more events than an LDL-C strategy, whereas an apoB strategy would prevent 500,000 more events than a non-HDL-C strategy. These examples emphasize the greater potential for using apoB rather than non-HDL-C and LDL-C.

100 Lipoproteins – Role in Health and Diseases

**4. ApoB versus non-HDL-C** 

not easily penetrate into the arterial wall.

they have learnt that the bad C is LDL-C.

LDL-C in recognizing the risk of CV disease and effects of statin therapy. Additional results (55) also favor apoB over LDL-C, and others are also reported in the section on the apo-ratio below. Such major studies are the AMORIS (3,44,56,57). In our study we found the steepest risk-relationship for MI with increasing values of apoB followed by non-HDL-C and the lowest increase in relation to LDL-C values with similar risk progressions for men and women **(Figure 2 and Figure 3 left).** Also in the INTERHEART (58,59) and ISIS-studies (60) as well as those summarized in the ERFC-meta-analyses (8,10) apoB was strongly related to risk of MI. In meta-analyses similar strong findings for apoB versus LDL-C are summarized by a large

ApoB indicates the number of atherogenic particles whereas non-HDL-C indicates the C mass from all atherogenic fractions like VLDL, IDL and the large buoyant LDL and the most atherogenic sdLDL fractions. But is apoB similar to or better than non-HDL-C in predicting risk? Although there is a similarity between apoB and non-HDL-C, they may have different metabolic fate and thus impact on risk. The rationale for using non-HDL-C is based on the fact that there is a close relationship between non-HDL-C and apoB values. Usually the correlation is about 0.80–0.85. However, correlation is not the same as concordance. In fact, two variables can be highly correlated but also be highly discordant, i.e. they do not correspond well. Either they are too high or too low compared with the other variable. Importantly, discordance produces major errors in the middle of the population distribution. Sniderman has frequently presented data with explanations of the advantages of apoB over LDL-C and non-HDL-C (4,13,14,21,62,63). Commonly, the sdLDL-particles contribute much to the large numbers of atherogenic particles, i.e. the apoB number is high and these small particles can easily penetrate into the arterial wall. However, in conditions with high non-HDL-C due to high VLDL-C and high large buoyant LDL-C the sdLDLparticles may be rather low in numbers indicating comparatively low numbers of apoB particles. These larger cholesterol-containing VLDL and IDL particles, although rich in C, do

Of interest, the number of apoB is more closely associated with insulin resistance or markers of the metabolic syndrome than either LDL-C or non-HDL-C (3,62,63). Thus, in patients with hypertriglyceridaemia with normal, or even low LDL-C values, i.e. patients with the metabolic syndrome (MetS), and in patients with overt diabetes, apoB has been shown to be superior to non-HDL-C in predicting vascular risk (3,62,63). Again, even if non-HDL-C and apoB correlate, they are not the same biologically or clinically. In most cases apoB is associated with a higher CV risk than non-HDL-C as well as LDL-C. Furthermore, non-HDL-C may not be that easy to understand or explain for the clinician or the patient once

Many studies and clinical trials have been published showing that apoB has a stronger capacity to identify all different phenotypes and to better predict CV risk than both LDL-C and non-HDL-C (3,11,61-63). Sniderman et al. (62) have published a convincing metaanalysis of results from published epidemiological studies that contains estimates of the

number of international scientists and clinicians in more detail (4,13,61,62).

However, in another major meta-analysis by Boekholdt et al. (64) they studied 62,154 patients enrolled in 8 statin trials published between 1994 and 2008. Among 38,153 statin treated patients 158 developed fatal MI, 1,678 non-fatal MI, 615 fatal events from other coronary artery disease, 2,806 hospitalizations for unstable angina, and 1,029 fatal or nonfatal strokes occurred during follow-up. The adjusted HRs for major CV events per 1-SD increase were 1.13 (95% CI, 1.10-1.17) for LDL-C, 1.16 (1.12- 1.19) for non-HDL-C, and 1.14 (1.11-1.18) for apoB. These HRs were significantly higher for non-HDL-C than LDL-C (p = 0.002) and apoB (p = 0.02). Thus, from both these meta-analyses non-HDL-C stands out as a stronger predictor of CV diseases than LDL-C. The explanation for the different findings of apoB in these two meta-analyses is unclear but may be explained by the fact that the first study is based on data from a prospective risk studies, whereas the second study reflects effects of statins on lipid and lipoprotein metabolism. Further comments are given in the discussion.

#### **5. Physiological and pathophysiological aspects of apoA-I**

There are many subgroups of particles of HDL with different lipid and apo compositions (3,12,29). Beyond apoA-I there are other apos such as apoA-II, apoA-III, apoC-III, apoD and apoM. ApoA-I is the major protein in HDL and this protein is taken to represent HDL metabolism since it occurs almost exclusively in HDL particles. By measuring HDL-C the amount of C transported in blood is indicated to represent the reverse cholesterol transport (RCT), a major protective aspect of HDL metabolism – by laymen named "the good cholesterol". ApoA-I initiates the RCT process in peripheral tissues. ApoA-I has also many other functions beyond RCT since apoA-I is involved in anti-inflammation, anti-oxidation, anti-infectious activity, anti-proteas activity, anti-apoptotic, and anti-thrombotic functions (3). Furthermore, apoA-I can initiate the endothelial production of nitric oxide that is of vital help in producing vasodilation (3). Furthermore apoA-I may help to regulate glucoseinsulin homeostasis. Thus, by measuring apoA-I you may get additional "protective" effects above those given only by the HDL-C number. For methodological reasons Warnick and others prefer to use apoA-I rather than HDL-C methods (19). HDL and apoA-I metabolism are reviewed in more detail, see ref. (3,12,29,65-67).

#### **5.1. Plasma levels of apoA-I and target values for therapy**

The plasma concentration of apoA-I can vary from 0.1 to over 3 g/L. Reference values have been published by Cantois et al. already in 1996 (37). There is little variation with fastingnon-fasting (68). Normally women have 0.1-0.3 g/L higher apoA-I values than men, similar to the higher HDL-C values for women. After menopause apoA-I values commonly decrease in parallel with HDL-C. However, there have been few published recommendations regarding what should be a "normal apoA-I value". A normal value for any adult should be at least close to 1 g/L or above. So far, there have been few recommendations on valid cut values indicating increased CV risk and target values. Values should be given in two decimals. For further comments, see the section on the apo-ratio.

#### **5.2. Biological variation of apoA-I**

ApoA-I and ApoA-II may also enter the cerebrospinal flow via the choroid plexus (69). Reduction in the HDL apoA-I/apoC-III ratio, changes in the HDL subpopulation distribution and an increase in HDL oxidation potential correlated with the development of MI in young patients (70). In a Korean study of 15,154 healthy subjects higher CRP levels were associated with significantly lower HDL-C and apoA-I levels, and also higher apoB values (71). In a US population of 8,708 apparently healthy population apoA-I was strongly positively associated, whereas apoB was significantly reduced with alcohol intake. Similarly the transaminases AST, ALT and gamma-GT increased with higher alcohol consumption (72).

#### **5.3. ApoA-I and risk for CV events**

Already in 1978,1979, Avogaro et al. (39,40) showed that apoA-I was as good as lipids in predicting myocardial infarction (MI) in those under 50 years of age but apoA-I was a better predictor in those over 60 years of age. In the Swedish APSIS study (73) Held et al. in 1994 studied patients with angina pectoris. During a median follow-up time of 3.3 years (2,663 patient years), 37 patients suffered a CV death, 30 suffered a non-fatal MI and 100 underwent a revascularization. Apo-I and TG were predictors of CV death or non-fatal MI in univariate analyses, but only apoA-I remained as an independent predictor in multivariate analyses. All lipid variables except LDL-C were related to the risk of revascularization in univariate analyses, but only apoA-I and apoB were independent predictors of such events. They concluded that apolipoprotein levels were better predictors of CV events than other lipid parameters in patients with stable angina pectoris.

Many studies have shown an inverse relationship between apoA-I and MI (3,44,74,52,53,59,60). In a study of Japanese Americans apoA-I predicted coronary heart disease only at low concentrations of HDL (75). High apoA-I values have been found to correlate with low risk for MI in AMORIS as indicated **(Figure 3, right).** Luc et al. also found that apoA-I is the best prospective risk marker of several other apoproteins in HDL (76). In the large INTERHEART case-control study apoA-I had a greater protective effect of MI at a wider range of apoA-I values than HDL-C (58,59). Patel et al. (77) found that ApoA-I levels are a consistent discriminator of atherosclerotic burden among patients with stable CAD.

102 Lipoproteins – Role in Health and Diseases

are reviewed in more detail, see ref. (3,12,29,65-67).

**5.2. Biological variation of apoA-I** 

**5.3. ApoA-I and risk for CV events** 

**5.1. Plasma levels of apoA-I and target values for therapy** 

ALT and gamma-GT increased with higher alcohol consumption (72).

above those given only by the HDL-C number. For methodological reasons Warnick and others prefer to use apoA-I rather than HDL-C methods (19). HDL and apoA-I metabolism

The plasma concentration of apoA-I can vary from 0.1 to over 3 g/L. Reference values have been published by Cantois et al. already in 1996 (37). There is little variation with fastingnon-fasting (68). Normally women have 0.1-0.3 g/L higher apoA-I values than men, similar to the higher HDL-C values for women. After menopause apoA-I values commonly decrease in parallel with HDL-C. However, there have been few published recommendations regarding what should be a "normal apoA-I value". A normal value for any adult should be at least close to 1 g/L or above. So far, there have been few recommendations on valid cut values indicating increased CV risk and target values. Values should be given in two decimals. For further comments, see the section on the apo-ratio.

ApoA-I and ApoA-II may also enter the cerebrospinal flow via the choroid plexus (69). Reduction in the HDL apoA-I/apoC-III ratio, changes in the HDL subpopulation distribution and an increase in HDL oxidation potential correlated with the development of MI in young patients (70). In a Korean study of 15,154 healthy subjects higher CRP levels were associated with significantly lower HDL-C and apoA-I levels, and also higher apoB values (71). In a US population of 8,708 apparently healthy population apoA-I was strongly positively associated, whereas apoB was significantly reduced with alcohol intake. Similarly the transaminases AST,

Already in 1978,1979, Avogaro et al. (39,40) showed that apoA-I was as good as lipids in predicting myocardial infarction (MI) in those under 50 years of age but apoA-I was a better predictor in those over 60 years of age. In the Swedish APSIS study (73) Held et al. in 1994 studied patients with angina pectoris. During a median follow-up time of 3.3 years (2,663 patient years), 37 patients suffered a CV death, 30 suffered a non-fatal MI and 100 underwent a revascularization. Apo-I and TG were predictors of CV death or non-fatal MI in univariate analyses, but only apoA-I remained as an independent predictor in multivariate analyses. All lipid variables except LDL-C were related to the risk of revascularization in univariate analyses, but only apoA-I and apoB were independent predictors of such events. They concluded that apolipoprotein levels were better predictors

Many studies have shown an inverse relationship between apoA-I and MI (3,44,74,52,53,59,60). In a study of Japanese Americans apoA-I predicted coronary heart disease only at low concentrations of HDL (75). High apoA-I values have been found to correlate with low risk for MI in AMORIS as indicated **(Figure 3, right).** Luc et al. also found

of CV events than other lipid parameters in patients with stable angina pectoris.

**Figure 3.** Left; The AMORIS study: Fatal myocardial infarction (Odds Ratio) is related to increasing values of apoB. The values are adjusted for age, TC and TG. Right; The AMORIS study: Fatal myocardial infarction is related to decreasing values of the apoA-I. The values are adjusted for age, gender, TC and TG. Similar pattern for men (män) and women (kvinnor).

In the CORONA study performed in patients with severe heart failure (placebo versus rosuvastatin) apoA-I, in univariate analysis, was the second best (after apoB plus apoA-I) of all different lipid fractions in predicting total death and MACE (MAjor Coronary Events). Furthermore, in a multivariate stepwise analysis apoA-I ranked fifth, better than high sensitivity CRP (hsCRP), of all 14 predictors of outcome where no conventional lipid fraction was significant. The best predictor was pro-BNP (78).

In a study of risk of stroke in Taiwan it was shown that apoA-I but not apoB levels may serve as an effect modifier of hypertension for the risk of stroke events (79).

In the combined analysis of data from the IDEAL statin trial and the Epic-Norfolk casecontrol study (80) very high HDL-C due to enlarged HDL-particles values were associated with increased rather than decreased CV risk. However, in contrast, apoA-I appears not to turn into a significant risk factor at high plasma concentrations. They conclude that apoA-I is associated with CHD risk independently from HDL size suggesting that the cardioprotective role of large HDL might be more closely related to its apoA-I content than to HDL size per se. These observations may have important consequences for future CAD risk assessment and novel treatment strategies. Indeed, several experimental studies have pointed to a crucial role for apoA-I in protection against atherosclerosis (3,12,65,81).

In the AFCAPS /TexCAPS statin trial (placebo versus lovastatin) multivariate analysis showed that apoA-I was better than HDL to predict outcome (52,53). In addition, the apo-ratio was the best of all lipids and apo-fractions to explain CV risk reduction, see further below.
