**2. Methodological pros and cons for using apoB, apoA-I and the apo-ratio versus conventional lipids**

### **2.1. Methodological problems for various lipids**

The most commonly used method world-wide to measure LDL-C is based on the Friedewald formula (16). However, errors are common and the methodological problems and shortcomings are not commonly recognized but have been discussed in many papers (17-25). Thus, the formula (LDL-C = TC – HDL-C – TG/5) is not valid for blood samples having triglycerides (TG) above 3.5-4 mmol/L, for patients with type III hyperlipoproteinemia or chylomicronemia or non-fasting specimens (17-19). The errors for LDL-C can be false positive in the range of 2-17% or false negative between 12-15% if TG levels are very low or closer to 4 mmol/L. This may create large problems for both clinicians and patients since patients may be misclassified as being at risk or not at risk according to guidelines. Similarly, it may be difficult for the clinician to evaluate if a patient has been adequately treated to the target of LDL-C. Newer so called ''direct LDL-C methods'' have been developed and they are homogeneous methods, that is, assays that do not require a preliminary separation step, such as ultracentrifugation, or manual manipulation of the sample for determining LDL-C (9,18-20). However, these methods, although standardized at a given laboratory, do not always correlate well over the whole range of lipid values, and they are not even internationally standardized like those for apolipoproteins.

The practical problems of measuring HDL-C are also of concern and correlation between various methods are sometimes even worse than those for LDL-C (18,19,26). Consequently, the values for non-HDL-C (TC minus HDL-C) may also be subject to large variations due to the errors mainly for measuring HDL-C. However, there is an advantage for non-HDL-C over LDL-C determined by the Friedewald formula since non-HDL-C is not subject to influence of non-fasting that may distort the TG levels and make it difficult to obtain a correct value for LDL-C (27). Furthermore, non-HDL-C contains C from all atherogenic fractions i.e. VLDL, IDL and various forms of LDL. Thus, non-HDL-C which indicates the total mass of C is more likely to reflect the variation of atherogenic particle set up for many patients with various genotypes and phenotypes. Such patients may have a greater chance to be correctly identified as risk individuals based on non-HDL-C, rather than to an imprecise measure of only LDL-C. For the interested reader of methods and concerns of validity, see further excellent reviews (18,19,26,27).

#### **2.2. Methodological advantages for apolipoproteins**

96 Lipoproteins – Role in Health and Diseases

in order to be recognized and used in clinical practice.

and various clinical complications like myocardial infarction (MI), stroke and other severe events to find out if there is evidence for using apoB and apoA-I, and especially the apoB/apoA-I ratio (apo-ratio) motivating clinical use of these risk markers/predictors. Both similarities, but mainly differences between apos and conventional lipids to predict CV risk, will be highlighted. Methodological aspects and the role of apoB and apoA-I, the two determinants of the apo-ratio, will first be commented. The major part of the paper describes the role of the apo-ratio as a CV risk marker/predictor. The overall conclusion from this paper will be that apoB, apoA-I and the apo-ratio merit to be included in future guidelines

**Figure 1.** The figure shows the atherogenic particles containing one apoB protein per VLDL, IDL, large buoyant LDL, small dense LDL particles and the anti-atherogenic lipoproteins containing apoA-I. The balance between apoB and apoA-I, i.e., the apoB/apoA-I ratio, reflects the balance between the "bad cholesterol particles and the good cholesterol particles". This apo-ratio is strongly related to

**2. Methodological pros and cons for using apoB, apoA-I and the apo-ratio** 

The most commonly used method world-wide to measure LDL-C is based on the Friedewald formula (16). However, errors are common and the methodological problems and shortcomings are not commonly recognized but have been discussed in many papers (17-25). Thus, the formula (LDL-C = TC – HDL-C – TG/5) is not valid for blood samples having triglycerides (TG) above 3.5-4 mmol/L, for patients with type III hyperlipoproteinemia or chylomicronemia or non-fasting specimens (17-19). The errors for

cardiovascular risk, the higher the ratio, the higher is the risk. (From reference 3).

**versus conventional lipids** 

**2.1. Methodological problems for various lipids** 

There are methodological advantages of using apoB and apoA-I compared to LDL-C and HDL-C since the apo-methods have been internationally standardized according to WHO-IFCC already in 1990-ies (26,28,29). The standardization initiatives for apo B have proceeded more quickly and more successfully than for LDL-C. The WHO-IFCC collaboration has resulted in the development of secondary reference material to ensure traceability of manufacturer calibrators to an approved standard. The bias and imprecision for 22 immunonephelometric and immunoturbidimetric assays ranged were usually below 5%. These errors are commonly smaller than that for calculated LDL-C and lipid ratios. Costs for measuring apos can be much reduced if apos are introduced as routine methods. However, pedagogical aspects (education of physicians, patients and laymen), and the well documented and cemented LDL-paradigm will make it difficult to convince guideline committees to introduce apoB and apoA-I as CV risk predictors. Importantly, this should not invalidate that apos are accepted as strong risk markers especially since so many other methods determining LDL-C and HDL-C are accepted in guidelines despite rather weak correlations between various methods due to incomplete standardizations.
