**16. Discussion**

Today, LDL-C, non-HDL-C and lipid ratios are prioritized in international guidelines although apoB has also been mentioned in a few guidelines (9,18,38,178-180). In this review evidence is given indicating that apoB, apoA-I, and especially the apo-ratio, are at least equally good, or often even better than conventional lipids to predict CV risk prospectively and during lipid-lowering treatment. Much of this new information has not yet been included in any previously published meta-analyses. It is therefore of importance to review these data obtained from countries in the whole world in order to get the full information of what apolipoproteins can deliver for CV risk prediction and evaluation. These findings and advantages are summarized below and also briefly in **Table 3 (I) and (II).** 

The biological relevance of using apoB and apoA-I as markers of CV risk is convincing since these proteins are carriers of lipids in the circulation and deliver C to peripheral tissues including the arterial wall (mediated by apoB). ApoA-I can remove C from the subendothelial space for breakdown and removal through the bile and further GI excretion. ApoA-I has also my other protective actions as summarized above and can thereby modify or inhibit inflammation and atherogenesis provoked by oxidation and modification of LDL-C with apoB (3,64). Thus, both apoB and apoA-I are biologically and patho-physiologically strongly active in normal biology and in plaque formation.

There are also methodological advantages of using these apos (3,23,28,65). Direct measurements of apoB and apoA-I by internationally standardized methods are available, analysis can be performed even if taken from patients in the non-fasted state, apos can be trustfully analyzed on frozen samples, the errors of the methods are not dependent on TG levels, and the methodological errors are usually low. Furthermore, costs for the direct analysis can be low as is the case in many countries. Importantly, the apo-ratio reflects the whole lipoprotein spectrum of virtually all phenotypes (type III patients need additional definition of risk) into one number. No sort like mg/dL or mmol/L has to be given for the ratio, which may otherwise be difficult to convert to understandable numbers in different countries. In summary, just one number of the apoB/apoA-I ratio indicates the risk level, similar numbers in all parts of the world. The higher the number, the higher is the risk (3) as also supported by a vast number of studies summarized in this paper. The cardiovascular risk line related to increasing value of the apo-ratio seems to be very similar world-wide (**Figure 10).**

ApoB, which indicates the number of potentially atherogenic particles, mainly sdLDL particles, has in a majority of publications been shown to be a better predictor of CV risk than LDL-C (9,11,13,14,44,59-62,88,89 and others) but in several instances apoB and non-HDL-C seem to indicate similar CV risk (10). One explanation why apoB may be better than non-HDL-C in risk prediction may be due to the fact that larger VLDL- and IDL-Ccontaining particles may have less potential to penetrate into the arterial walls than

**16. Discussion** 

(**Figure 10).**

apoB by 5-10 % at the most and the apo-ratio by about 5% and increase apoA-I by about 5%.

Today, LDL-C, non-HDL-C and lipid ratios are prioritized in international guidelines although apoB has also been mentioned in a few guidelines (9,18,38,178-180). In this review evidence is given indicating that apoB, apoA-I, and especially the apo-ratio, are at least equally good, or often even better than conventional lipids to predict CV risk prospectively and during lipid-lowering treatment. Much of this new information has not yet been included in any previously published meta-analyses. It is therefore of importance to review these data obtained from countries in the whole world in order to get the full information of what apolipoproteins can deliver for CV risk prediction and evaluation. These findings and

The biological relevance of using apoB and apoA-I as markers of CV risk is convincing since these proteins are carriers of lipids in the circulation and deliver C to peripheral tissues including the arterial wall (mediated by apoB). ApoA-I can remove C from the subendothelial space for breakdown and removal through the bile and further GI excretion. ApoA-I has also my other protective actions as summarized above and can thereby modify or inhibit inflammation and atherogenesis provoked by oxidation and modification of LDL-C with apoB (3,64). Thus, both apoB and apoA-I are biologically and patho-physiologically

There are also methodological advantages of using these apos (3,23,28,65). Direct measurements of apoB and apoA-I by internationally standardized methods are available, analysis can be performed even if taken from patients in the non-fasted state, apos can be trustfully analyzed on frozen samples, the errors of the methods are not dependent on TG levels, and the methodological errors are usually low. Furthermore, costs for the direct analysis can be low as is the case in many countries. Importantly, the apo-ratio reflects the whole lipoprotein spectrum of virtually all phenotypes (type III patients need additional definition of risk) into one number. No sort like mg/dL or mmol/L has to be given for the ratio, which may otherwise be difficult to convert to understandable numbers in different countries. In summary, just one number of the apoB/apoA-I ratio indicates the risk level, similar numbers in all parts of the world. The higher the number, the higher is the risk (3) as also supported by a vast number of studies summarized in this paper. The cardiovascular risk line related to increasing value of the apo-ratio seems to be very similar world-wide

ApoB, which indicates the number of potentially atherogenic particles, mainly sdLDL particles, has in a majority of publications been shown to be a better predictor of CV risk than LDL-C (9,11,13,14,44,59-62,88,89 and others) but in several instances apoB and non-HDL-C seem to indicate similar CV risk (10). One explanation why apoB may be better than non-HDL-C in risk prediction may be due to the fact that larger VLDL- and IDL-C-

containing particles may have less potential to penetrate into the arterial walls than

For more information see reference 3 and data on effects of statins in section 13 above.

advantages are summarized below and also briefly in **Table 3 (I) and (II).** 

strongly active in normal biology and in plaque formation.

Table 3 (I) \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

#### **Relations to CV risk factors**

Strong associations with abdominal obesity, metabolic syndrome and both diabetes

#### **Relations to CV risk in univariate and multivariate analyses**

Risk relationships for individual apos ant the apo-ratio commonly remained after adjustment for multiple conventional risk factors.

#### **Relations to lipids and lipoproteins as predictors of CV risk**

Lipids and lipid-based ratios are rarely significantly better than apos or the apo-ratio. However, apos and the apo-ratio are at least as good as lipids and lipid ratios, but commonly significantly better than lipids to predict CV risk

#### **Relations to atherosclerosis**

Strong associations with atherosclerosis in carotid arteries (IMT), coronary atherosclerosis (angiography and IVUS), femoral plaques and impaired endothelial function. Predicts progression and regression of carotid and coronary atherosclerosis

#### **Relations to lipid-lowering treatment**

Predicts outcome in statin trials equally well or commonly better than conventional lipids and lipoproteins

Table 3 (II)

**Table 3.** (I) and (II). Summary of findings supporting the use of apoB, apoA-I and the apo-ratio.

sdLDL particles. In fact, in a number of large publications including meta-analyses apoB has been shown to be a stronger predictor than the next best predictor non-HDL-C (9,11,13,14,61,62). LDL-C is only the third best predictor of future CV risk according to major analyses (9,62) and so also during statin treatment (169-171). However, in another metaanalysis they found non-HDL-C to be better than LDL-C and apoB during statin treatment (64). In a majority of these studies data have been adjusted for age and gender as well as other confounding risk factors like blood pressure, smoking, obesity and commonly also diabetes and other lipids.

Direct comparative data of HDL-C versus apoA-I is more sparse and is still much debated due to the complexity of HDL metabolism. ApoA-I has often similar predictive value as HDL-C as presented in the ERFC meta-analysis (10). However, especially in the large INTERHEART study, apoA-I over the whole range of HDL-values was a better predictor of risk than HDL-C (59). Similarly, in the AFCAPS/TexCAPS statin study apoA-I was a stronger determinant of risk than HDL-C (52,53).

What about the lipid-ratios versus the apo-ratio, which has strongest relations to CV risk? Importantly, all ratios and especially the apo-ratio predict prospective risk better than any single lipid variable (3,4,7,22,44,52,53,56,59,60,80,88,89,100,101,169-171 and others). Similarly, during statin treatment ratios also beat single lipoproteins in predicting risk (168- 171,174). That should be obvious since ratios has a greater potential to find subjects at risk in whom the anti-atherogenic capacity of HDL-C or apoA-I are deranged. These data are also obtained when controlling for confounders i.e. conventional risk factors. Thus, the apo-ratio may have a better potential to identify subjects with different phenotypes than a single lipoprotein fraction. These strong findings in favor of any ratio, especially the apo-ratio, are strangely enough, not considered important in any international guideline despite the fact that ratios virtually in all studies in which ratios have been used outperform the results obtained by single lipoprotein fractions. Why this unscientific approach by guidelines committees?

Results from meta-analyses are generally well trusted but can also be questioned regarding selection criteria for including studies, acceptance of analytical and diagnostic methods used in each of the studies, primary and secondary variables used as major endpoints as well as the general conclusions drawn from the analyses. The results from the first ERFC metaanalysis have been taken to indicate that the apo-ratio and the TC/HDL-C ratio are equally good predictors of risk (10) and that apoB is equally good as risk predictor as non-HDL-C and apoA-I is equally predictive as HDL-C. The authors also open for future use of apolipoproteins especially in evaluation of risk of MI. In these risk conditions they found that apoB and apoA-I could be more useful in men than women, and in subjects with high TC, in those with low HDL-C, in individuals with hypertension, and in those with intermediate CV risk (Framingham risk score) apos can also be useful. They also found that the apo-ratio was a better predictor of CHD than stroke. However, in a recent publications in JAMA they conclude that the TC/HDL-C ratio had stronger predictive power than the apo-ratio when these ratios were added to conventional risk factors.

Two major critical views against these JAMA (see footnotes a/ and b/ below) papers may be raised that unfavorably affect the trust of using apos as risk predictors. Many early studies on apos included in these meta-analyses had large methodological errors (not internationally standardized) which may affect the conclusions on the credibility to use apos as risk predictors. This is unfair to the modern apo-technology which has much lower methodological errors.

130 Lipoproteins – Role in Health and Diseases

diabetes and other lipids.

committees?

stronger determinant of risk than HDL-C (52,53).

sdLDL particles. In fact, in a number of large publications including meta-analyses apoB has been shown to be a stronger predictor than the next best predictor non-HDL-C (9,11,13,14,61,62). LDL-C is only the third best predictor of future CV risk according to major analyses (9,62) and so also during statin treatment (169-171). However, in another metaanalysis they found non-HDL-C to be better than LDL-C and apoB during statin treatment (64). In a majority of these studies data have been adjusted for age and gender as well as other confounding risk factors like blood pressure, smoking, obesity and commonly also

Direct comparative data of HDL-C versus apoA-I is more sparse and is still much debated due to the complexity of HDL metabolism. ApoA-I has often similar predictive value as HDL-C as presented in the ERFC meta-analysis (10). However, especially in the large INTERHEART study, apoA-I over the whole range of HDL-values was a better predictor of risk than HDL-C (59). Similarly, in the AFCAPS/TexCAPS statin study apoA-I was a

What about the lipid-ratios versus the apo-ratio, which has strongest relations to CV risk? Importantly, all ratios and especially the apo-ratio predict prospective risk better than any single lipid variable (3,4,7,22,44,52,53,56,59,60,80,88,89,100,101,169-171 and others). Similarly, during statin treatment ratios also beat single lipoproteins in predicting risk (168- 171,174). That should be obvious since ratios has a greater potential to find subjects at risk in whom the anti-atherogenic capacity of HDL-C or apoA-I are deranged. These data are also obtained when controlling for confounders i.e. conventional risk factors. Thus, the apo-ratio may have a better potential to identify subjects with different phenotypes than a single lipoprotein fraction. These strong findings in favor of any ratio, especially the apo-ratio, are strangely enough, not considered important in any international guideline despite the fact that ratios virtually in all studies in which ratios have been used outperform the results obtained by single lipoprotein fractions. Why this unscientific approach by guidelines

Results from meta-analyses are generally well trusted but can also be questioned regarding selection criteria for including studies, acceptance of analytical and diagnostic methods used in each of the studies, primary and secondary variables used as major endpoints as well as the general conclusions drawn from the analyses. The results from the first ERFC metaanalysis have been taken to indicate that the apo-ratio and the TC/HDL-C ratio are equally good predictors of risk (10) and that apoB is equally good as risk predictor as non-HDL-C and apoA-I is equally predictive as HDL-C. The authors also open for future use of apolipoproteins especially in evaluation of risk of MI. In these risk conditions they found that apoB and apoA-I could be more useful in men than women, and in subjects with high TC, in those with low HDL-C, in individuals with hypertension, and in those with intermediate CV risk (Framingham risk score) apos can also be useful. They also found that the apo-ratio was a better predictor of CHD than stroke. However, in a recent publications in JAMA they conclude that the TC/HDL-C ratio had stronger predictive power than the

apo-ratio when these ratios were added to conventional risk factors.

Furthermore, in the ERFC studies they pooled non-fatal MI, all CHD fatalities, peripheral vascular diseases, and even all strokes, especially haemorrhagic stroke and unidentified stroke in very old people, into the primary variable "cardiovascular events". Such pooling of events considerably dilute the potential of adequate information yielded by an appropriately measured apos and the apo-ratio. This is especially the case for patients with risk of MI and in those suffering ischemic strokes in which positive diagnostic values have been obtained for apos as summarized in previously commented studies.

The authors also discuss some potential problems with introduction of apos such as need for education, lack of availability of apo-methods in the most laboratories, standardization problems as well as additional costs for such methods. All these aspects and possible problems must obviously be considered when new diagnostic tests shall be introduced for clinical use in risk evaluation. Yes, education is mandatory and may take time, but such problems must not over shade the importance of innovation of analytical tools. Costs can be significantly reduced if apo-tests become standard analyses. In fact, many biochemists already now favor these analyses over conventional lipids as documented previously in this paper.

Another criticism of the ERFC-studies is related to which studies were excluded (lack of confounding variables or case-control studies) from the meta-analyses in ERFC. Thus, major studies like AMORIS (44), INTERHEART (58) and ISIS (60) were not included in the ERFC meta-analysis. Neither were their positive results commented in the discussion on risk of MI despite the fact that these three studies have six times as many well defined events than those in the ERFC studies. In all these large studies apoB, apoA-I and especially the aporatio, due to their large number of events, were each significantly stronger predictors than conventional lipids. In ERFC there were many studies, but few of these studies showed significant differences between lipids and apos due to few well defined hard events. Neither did ERFC point out that the apo-ratio also seems to be the best variable to describe the remaining CV risk after statin treatment. This has been shown especially in the statin trials like AFCAPS/TexCAPS (52,53), IDEAL (169-171), TNT (168-170), CARDS (181), and JUPITER (176) as well as in studies on regression of atherosclerosis during lipid-lowering therapy (172-174) as pointed out above. In most of these studies the data were also adjusted for age, gender, conventional lipids and lipoproteins as well as other major risk factors like blood pressure, smoking, obesity and diabetes. Grundy simply concludes in the JAMA editorial

a/ The Emerging Risk Factors Collaboration. Lipid-Related Markers and Cardiovascular Disease Prediction. JAMA. 2012;307(23):2499-2506.

b/ Scott M. Grundy. Editorial: Use of Emerging Lipoprotein Risk Factors in Assessment of Cardiovascular Risk. 2012;307(23):2540-2541.

that conventional risk factors plus LDL-C, and possibly one more risk factor, is enough as tools for prediction of risk – a very conservative approach which is so (too!) common in US!

How much do confounders/risk factors impact on the results from all these CV risk studies? Importantly, in ERFC (10) and also in the majority of studies cited above, the impact of adjusting for major confounders was very small and only changed the risk (HR, RR or OR) to a minor degree. In fact, the apolipoproteins added value, measured as net reclassification index (NRI) in several large studies (88,91,170). This indicates that apolipoproteins, especially the apo-ratio, could change the numbers of individuals either to a higher or a lower CV risk compared to conventional lipids. Newly developed risk algorithms based on the apo-ratio have also been developed showing at least equal predictive or even better values than conventional risk algorithms (102,117,118). Thus, apoB, apoA-I and the apo-ratio can already now be used in clinical settings.

In the present review the apo-ratio has been shown to be closely related to many different types of CV events in prospective studies. These common diseases are myocardial infarction, stroke, especially ischemic stroke, heart failure, renal failure, aortic aneurysms, development of diabetes, including retinopathy **(Table 3 I and II)**. However, in the metaanalyses published so far only CV events have been chosen as endpoints and other manifestation of CV risk related to atherosclerosis have been excluded.

Is the apo-ratio useful in predicting various metabolic and inflammatory conditions commonly underlying atherosclerosis and its future consequences? In fact, the apo-ratio has also been found to be a valuable summarizing index of lipid-abnormalities and their complications in a large number of studies of the MetS and/or diabetes (125-139,143,144). In addition, the apo-ratio values are also increased in patients with hypertension, obesity, in pubertal children and in those with heredity for CV diseases (130,141). The apo-ratio is also more closely than lipids related to atherosclerosis in a large number of studies in which different techniques like coronary angiography, arterial wall thickness obtained by ultrasound techniques in the carotid arteries (CIMT values) or even in the coronary arteries by intravascular ultrasound (IVUS) and arterial abnormalities such as the endothelial dysfunction have been used (145-162). Thus, in all these disease or risk situations the aporatio may identify those at an increased risk even better than what is currently performed by using LDL-C or the recently recommended non-HDL-C.

The newest research data on the apo-ratio have not yet been reviewed by international guideline committees. Thus, so far, in the newest guidelines developed over the last few years non-HDL-C and apoB are mentioned, and accepted for clinical use, whereas the aporatio is still waiting for acceptance (9,35,178,179).

In conclusion; with all the new knowledge presented in this paper about the strong relations between apoB, apoA-I, and the apo-ratio, and CV risk as well as other disease manifestations, it is proposed, as many researchers have already done, that these strong risk predictors/factors/markers are included in new guidelines. In many disease conditions and manifestations of atherosclerosis apolipoproteins are at least equally informative, and often better than LDL-C, non-HDL-C and lipid ratios in predicting risk. It is realized that there will be pedagogical hurdles, but it should be possible to educate physicians, patients and health providers to understand that these apolipoproteins are markers of normal and abnormal cholesterol metabolism. The apo-ratio simply reflects the "balance between the bad cholesterols and the good cholesterols" technically measured by apolipoproteins. The apo-ratio is a valid cardiovascular risk index (CRI) that reflects the level of CV risk for virtually all patients with different lipid phenotypes, the higher the value of the apo-ratio, the higher is the risk. Finally, targeting lower values (about 0.50) of the apo-ratio during therapy may more correctly identify who is at risk or not at risk, and how high is the risk? Does the risk depend on the atherogenic apoB, or the anti-atherogenic apoA-I or rather on the most informative value i.e. the apo-ratio which summarizes the level of risk in a simple way? Since physicians usually only manage to effectively evaluate and trust one laboratory marker, the apo-ratio is such a valid marker. By simply plotting the value for a given patient on the risk line you can easily follow improvement during therapy and also motivate the patient to improve values to normal levels **(Figure 10**). New guidelines should at least contain equally objective information (cut-values and target values) on how to use apoB, apoA-I, and the apo-ratio as on lipids so that physicians can choose whichever diagnostic marker of risk they prefer. Gradually this new apolipoprotein-based risk classification with a focus on the apoB/apoA-I ratio may, or rather should be, introduced in clinical practice.
