**7. Prospective cardiovascular risk studies – Relations to apoB, apoA-I and the apoB/apoA-I-ratio**

#### **7.1. The AMORIS prospective study and risk of myocardial infarction (MI)**

The Swedish AMORIS (Apolipoprotein-related MOrtality RISk) study is the largest of all studies in which apoB and apoA-I have been measured in more than 175.000 individuals followed prospectively for up to 25 years. The participating subjects were recruited from health check-ups during 1985-1996. Their age ranged from below 10 years to above 90 years. In these years health screening was very common in Sweden. Subjects included in the database called AMORIS were mainly healthy, not acutely ill or hospitalized and no subject participated in clinical trials. They were all treated by their general practitioners in the greater Stockholm area and they constitute a valid socio-economical cohort of the greater Stockholm population as indicated in several of our papers presented below. Large blood screening programs were used including some 8,000 determinations of LDL-C according to Friedewald. Simultaneously apoB and apoA-I were analyzed by automated immunoturbidimetric methods in all 175.000 subjects according to the WHO-IFCC protocol and in collaboration with their representatives (82). LDL-C was calculated according to the Jungner formula (44). The Jungner formula yields the same LDL-C values as those obtained by using the Friedewald formula (16) as confirmed by Talmud et al. (47). Also HDL-C values were determined by the Friedewald formula once LDL-C was calculated by the Jungner formula. All analyses in the AMORIS laboratory database (confounding clinical risk factors like hypertension, diabetes and obesity were available in cohorts) were performed by automated methods at the same CALAB laboratory headed by Ingmar Jungner. Several papers were published describing the apoB, apoA-I and the apo-ratio characteristics of the population and the methods (3,21,44,82-85). In an early AMORIS study we have previously noted that patients with type IIB dyslipidemias, i.e. combined hypercholesterolemia and hypertriglyceridemia, had the highest apo-ratio (86). The subsequent CV manifestations were related to the laboratory variables obtained at the first visit to the physician.

104 Lipoproteins – Role in Health and Diseases

**of risk** 

**6. General comments on the validity of using a ratio as a primary marker** 

Lipid and lipoprotein ratios like TC/HDL-C and LDL-C/HDL-C have been used in various international guidelines for decades to define CV risk. However, LDL-C has in the vast number of guidelines dominated as the primary risk marker why ratios rarely are used today in clinical practice. One major reason why the lipid ratios are questioned as relevant risk markers is due to the fact that HDL-C is included in the value for TC, so HDL-C occurs both in the nominator and denominator of the ratio. Similarly, since LDL-C most commonly is derived by the Friedewald formula, HDL-C is involved as a factor for calculating LDL-C and therefore also indirectly in the nominator and denominator of that ratio. Therefore physicians are hesitant to the mathematical way of dividing various lipid numbers to obtain a mathematical, but, in their mind, not a biologically relevant ratio. When so called direct methods are used for measuring LDL-C this problem is less. In recent years non-HDL-C has been recommended as the next primary risk variable and the new non-HDL-C/HDL-C ratio has been defined. Interestingly, this ratio gives the same final number of risk as that of the TC/HDL-C ratio.

Most researchers and guidelines recommend the use the TC/HDL-C ratio since calculation of this ratio is not dependent on that blood sampling has been performed in the fasted state. This is the same argument as for using non-HDL-C rather than calculated LDL-C. The challenge now is can the apo-ratio, which summarizes the CV risk related to all atherogenic and all anti-atherogenic variables into one number, be the next rational choice as a primary risk variable? Does the apo-ratio add to information already obtained by lipids and lipid ratios? And are the values for apoB, apoA-I and especially the apo-ratio much influenced by other confounding risk factors? These and many other questions are addressed in the

**7. Prospective cardiovascular risk studies – Relations to apoB, apoA-I and** 

The Swedish AMORIS (Apolipoprotein-related MOrtality RISk) study is the largest of all studies in which apoB and apoA-I have been measured in more than 175.000 individuals followed prospectively for up to 25 years. The participating subjects were recruited from health check-ups during 1985-1996. Their age ranged from below 10 years to above 90 years. In these years health screening was very common in Sweden. Subjects included in the database called AMORIS were mainly healthy, not acutely ill or hospitalized and no subject participated in clinical trials. They were all treated by their general practitioners in the greater Stockholm area and they constitute a valid socio-economical cohort of the greater Stockholm population as indicated in several of our papers presented below. Large blood screening programs were used including some 8,000 determinations of LDL-C according to Friedewald. Simultaneously apoB and apoA-I were analyzed by automated immunoturbidimetric methods in all 175.000 subjects according to the WHO-IFCC protocol and in collaboration with their representatives (82). LDL-C was calculated according to the

**7.1. The AMORIS prospective study and risk of myocardial infarction (MI)** 

sections below based on a vast number of publications.

**the apoB/apoA-I-ratio** 

In 2001 we presented the first endpoint paper based on 98,722 men and 76,831 women in the Lancet (44). We found a strong direct relationship between apoB and an indirect inverse relationship between apoA-I and risk of MI (men = 864, women = 359) **(Figure 3)**. Furthermore, apoB was a stronger risk factor than LDL-C especially at low values of LDL-C. The apo-ratio was the strongest lipid-related factor **(Figure 4).** In the left part of the figure the values for apoB and apoA-I divided into quartiles are displayed in a three dimensional way. Thus, in those with highest values of apoB and in those with lowest apoA-I values the risk increased about 6-fold in a stepwise fashion compared to those with lowest apoB and highest apoA-I values. The highly significant results were similar for men and women and remained after adjusting for age, TC and TG. The figure clearly illustrate that the risk is about the same for those with an increased apoB at highest apoA-I levels, as the risk for those with lowest apoA-I levels but with low apoB values. Thus the figure illustrates the importance of measuring both apoB and apoA-I to get correct information on MI risk level. In the right part of the figure the same results can also be depicted as a straight line (semi-log scale) showing the impact of higher apo-ratio versus increased risk of MI. We also found that apoB was significantly better to predict risk than LDL-C especially for those with low values for LDL-C.

**Figure 4.** Left; The AMORIS study: Fatal myocardial infarction (Risk ratio) is related to increasing values of apoB and decreasing values of apoA-I. The values are adjusted for age, TC and TG. Similar pattern is seen for men and women (reference 3). Right; The AMORIS study: Fatal myocardial infarction is related to increasing values of the apoB/apoA-I ratio. The values are adjusted for age, gender, TC and TG. (Both figures from reference 3).

With increasing values of the apo-ratio there was a parallel increase in apoB, LDL-C, non-HDL-C and TG **(Figure 5, left)** and a decrease in apoA-I and HDL-C values **(Figure 5, right).** This figure illustrates that an increasing apo-ratio indirectly also indicate the contribution of the other lipids as risk factors. In multivariate analyses the apo-ratio is the strongest of all lipid-related variables and is thus the best summarizing risk variable.

**Figure 5.** Left; the apo-ratio in deciles (x-axis) versus different atherogenic lipid fractions (y-axis). Right; the apo-ratio in deciles (x-axis) versus values for HDL-C and apoA-I (y-axis). Both figures from the AMORIS study (in reference 3).

In collaboration with Sniderman we have also published data from AMORIS showing that the apo-ratio has a significantly stronger relation with MI than any other lipid-based ratio (3,4,21).

In another AMORIS cohort including 69,029 men and 57,167 women who were followed for a mean of 10.3 years we determined LDL size as reflected by the LDL-C/apoB ratio (87). Because LDL size did not add predictive information to the apo-ratio, it appears that this apo-ratio also captures the risk related to LDL size. These findings add to our previously published results from AMORIS that indicates that the apo-ratio is the best single lipidrelated summary index of risk and that TC, TG, non-HDL-C, and LDL-C do not add significant predictive power to the apo-ratio.

#### **7.2. The apo-ratio and inflammatory risk factors – relations to MI, stroke and heart failure in the AMORIS study**

The risk relation to age during prolonged follow-up was also studied in an AMORIS population (n = 149,121) free of previous MI at blood sampling. They were followed from 1985 to 2002 with respect to n = 6,794 first cases of MI. The mean value of the apo-ratio for men was 1.0 and for women 0.85 at baseline. In collaboration with Holme we found that the apo-ratio was somewhat stronger for those developing non-fatal than fatal MI (88). The risk was also stronger associated with the apo-ratio in those < 65 years of age than above, but risk remained significantly related to the apo-ratio also in the older population. In multivariate analyses the apo-ratio was a better predictor than TCHDL-C. Furthermore, the apo-ratio added clinically significant information to TCHDL-C in men as reflected by a net reclassification improvement (NRI) of 9.4% (P < 0.0001). Furthermore, also in patients developing heart failure, a common complication after MI, the apo-ratio is the best lipidrelated variable to classify risk especially in men (89).

106 Lipoproteins – Role in Health and Diseases

AMORIS study (in reference 3).

significant predictive power to the apo-ratio.

**heart failure in the AMORIS study** 

With increasing values of the apo-ratio there was a parallel increase in apoB, LDL-C, non-HDL-C and TG **(Figure 5, left)** and a decrease in apoA-I and HDL-C values **(Figure 5, right).** This figure illustrates that an increasing apo-ratio indirectly also indicate the contribution of the other lipids as risk factors. In multivariate analyses the apo-ratio is the strongest of all

**Figure 5.** Left; the apo-ratio in deciles (x-axis) versus different atherogenic lipid fractions (y-axis). Right; the apo-ratio in deciles (x-axis) versus values for HDL-C and apoA-I (y-axis). Both figures from the

In collaboration with Sniderman we have also published data from AMORIS showing that the apo-ratio has a significantly stronger relation with MI than any other lipid-based ratio (3,4,21). In another AMORIS cohort including 69,029 men and 57,167 women who were followed for a mean of 10.3 years we determined LDL size as reflected by the LDL-C/apoB ratio (87). Because LDL size did not add predictive information to the apo-ratio, it appears that this apo-ratio also captures the risk related to LDL size. These findings add to our previously published results from AMORIS that indicates that the apo-ratio is the best single lipidrelated summary index of risk and that TC, TG, non-HDL-C, and LDL-C do not add

**7.2. The apo-ratio and inflammatory risk factors – relations to MI, stroke and** 

The risk relation to age during prolonged follow-up was also studied in an AMORIS population (n = 149,121) free of previous MI at blood sampling. They were followed from 1985 to 2002 with respect to n = 6,794 first cases of MI. The mean value of the apo-ratio for men was 1.0 and for women 0.85 at baseline. In collaboration with Holme we found that the apo-ratio was somewhat stronger for those developing non-fatal than fatal MI (88). The risk was also stronger associated with the apo-ratio in those < 65 years of age than above, but

lipid-related variables and is thus the best summarizing risk variable.

Subsequently we have shown that for the inflammation marker haptoglobin (Hp) has strong relations with MI, stroke and heart failure in the AMORIS cohort (90). There were 11,216 men and 4,291 women who had a first MI, 8,463 men and 6,072 women who had a first stroke, and 4,670 and 3,634 who had a first heart failure, respectively. Based on 4,254 MI cases the risk of MI was about 4.5 times higher in the upper joint quartile of the apo-ratio as compared to the lower, whereas this relative risk for Hp was about 4.1. However, the attributable risk for the apo-ratio is higher since more subjects were classified into the top joint quartile of TC and the apo-ratio (12.8%) than that of TC and Hp (8.8%) and into the lower joint quartiles (12.1%) and (6.4%), respectively.

In another AMORIS-based cohort of 65,050 subjects Holme et al. (91) developed an inflammatory score comprising white blood cell count, haptoglobin and in a subgroup also CRP. After 11.8 years follow-up 3,649 MI, 2,663 stroke, 2,690 heart failure, in total 7, 456 MACE, occurred. In multivariate Cox proportional hazards analysis the inflammatory scores added predictive information over and above classical lipids such as TC and TG. Based on the apo-ratio, which was a stronger marker of CVD risk than conventional lipids, the inflammatory score added significant information value measured by net reclassification improvement, especially for those with the higher values for these variables. However, there was no statistically significant biological interaction between lipoproteins and the inflammatory markers. These data indicate that routinely used markers of inflammation in combination with the apo-ratio could be used in daily medical practice to assess CV risk.

We have also published data of lipid- and the apo-ratio from three cultures (Sweden, Iran, US) showing that the apo-ratio is highest in the Swedes (the AMORIS cohort) but similar in the Americans (NHANES) and Iranians (92). By contrast, the TC/HDL-C ratio is highest in the Iranians, intermediate in the Americans and lowest in the Swedes. There were similar associations of the pro-atherogenic and anti-atherogenic lipoproteins between the genders and variation with age in these three different cultures. These data indicate that complete characterization of lipoproteins requires measurement of apoB and apoA-I as well as lipoprotein lipids.
