Introduction

Cardiovascular disease is the most common cause of death worldwide. For the past many years total lipid profile i.e. total cholesterol, triglycerides, VLDL cholesterol and LDL cholesterol have been positively associated with coronary artery disease (CAD) while HDL cholesterol has inverse relation ₁,₂ But lipid and lipoprotein cholesterol levels have failed to explain the increased prevalence of CAD in normolipidemic patients₃. Recently serum apoA1 and apo B have been proposed as relatively better markers for assessing the risk of CAD and its treatment regimen. ApoA1 is the constituent component of HDL and apoB is of LDL. It was shown in the AMORIS study that apoA1 and apoB might be of greatest value in predicting the risk of myocardial infarction especially in patients having low or normal LDL cholesterol level ₄. A few countries like Canada have incorporated apoB in their clinical management guidelines ₅. Age and sex are the two important factors seemed to affect lipid levels. Studies regarding age and sex related variations in serum apoA1 and apoB levels are few and are only confined to European, North American and few Australian and African populations ₆,₇,₈. Such data is very limited in Indian population, which has a very high incidence of coronary artery disease. Global burden of CAD is mainly concentrated in developing countries ₉. These apolipoproteins may act as important CAD risk indicators in our population. Hence the present study was aimed to assess age and sex related variations in serum apoA1 and B levels in normal individuals and to investigate the role of these apolipoproteins as CAD risk indicators in Indian (Punjabi) population.

Material And Methods

In the present study two hundred and ninety patients (290) suffering from coronary artery disease (CAD) and 270 age and sex matched normal individuals were included. Patients were taken from the wards and OPDs of Guru Nanak Dev and ESI hospitals, Amritsar. Diagnosis of CAD was done by the treating experts on the basis of clinical symptoms, ECG changes and stress test. Patients were also subjected to angiography if required. CAD patients were also having some of the associated risk factors such as diabetes mellitus; hypertension etc and they were on the requisite treatment. Exclusion criteria included patients on lipid lowering drugs, having renal or thyroid disease and women having hysterectomy or on oral contraceptives or taking HRT. Normal individuals free from any evident symptom of the disease were taken as controls. They were subjected to ECG and stress test wherever possible to confirm their clinical state. Mean BMI of normal subjects was 26±3Kg/m² and of CAD patients was 27±4Kg/m².The control group represents the free living sample population and no selective criteria was included such as dietary restriction, increased/decreased physical activity or alcohol consumption or smoking status. Informed consent was taken from all the subjects included in the study. Both normal individuals and CAD patients were divided into 3 age groups: Group I (35-44 yrs), Group II (45-54 yrs) and Group III (55-64 yrs). Blood samples were taken after 12 hour overnight fast and serum was used for various investigations. Apolipoprotein A1 and B levels were estimated by immunoturbidemetric method ₇ using kits from Diasys India Pvt Ltd. Reagents and calibrators were in accordance with WHO International reference material for these apolipoproteins. It has been reported that fasting/no fasting conditions do not affect apolipoprotein levels. ₇ but fasting blood samples were preferred to avoid turbidity due to chylomicrons which could affect lipid levels. Total cholesterol levels were estimated by Zlatkis method as modified by Zak ₁₀. Triglycerides were estimated with enzymatic method from commercially available kits. HDL cholesterol was estimated after precipitating VLDL and LDL by using magnesium chloride and sodium phosphotungstate ₁₁. LDL cholesterol levels were estimated using the Friedwald's formula. ₁₂

Statistical analysis

Results

In normal males serum apoB levels tend to increase continuously from age group I to group III (Fig I). No in between decline in serum apoB levels was observed. Females also showed continuous increase in serum apoB levels with advancing age. But increase in serum apoB levels in males in each age group was relatively more as compared to females leading to significant sex related differences in apoB levels. Similar age and sex related variations were observed in serum LDL cholesterol levels in males and females (Fig II). A significant positive coefficient of correlation (r 0.68; p<0.01) was observed between serum apoB levels and LDL cholesterol levels. Age related variations in serum apoA1 levels were not similar to that of serum apoB levels. Serum apoA1 levels showed slight decrease in serum apoA1 levels after the age of 44 years in normal males and the levels continued to decrease till the age of 64 years (Fig I). No in between increase in serum apoA1 levels was observed in normal males. Females showed significant increase (p<0.01) in serum apoA1 levels after the age of 44 years. Then the levels declined after the age of 54 years but this decline was relatively earlier in males. No plateau in serum apoA1 levels was observed. In every age group females were having significantly raised (p<0.01) serum apoA1 levels as compared to males. Similar age and sex related variations were also observed in HDL cholesterol levels in normal males and females (Fig II). A significant positive coefficient of correlation (r 0.71; p<0.01) was observed between serum apoA1 and HDL cholesterol levels. The comparison between control group (males and females) and CAD patients (males and females) revealed that all parameters showed significant difference between the two populations (Table I). CAD patients were having raised apoB, apoB/apoA1 ratio, LDL cholesterol and triglycerides and low apoA1 and HDL cholesterol levels compared to control group.

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Discussion

Apolipoprotein B is a constituent component of LDL and apoA1 of HDL. Recently they have been proposed as better markers to assess the risk of coronary artery disease. Some workers have assessed the role of these apolipoproteins in coronary artery disease but the conclusions drawn are variable ₁₃,₁₄. Age and sex are two non modifiable risk factors of CAD which may affect serum apolipoprotein levels. Data regarding the effect of age and sex on serum apoA1 and apoB levels is very limited in Indian population. These uncertainties are limiting the use of these apolipoproteins in routine diagnosis, which may prove beneficial in our population having high incidence of coronary artery disease. To investigate their role in CAD, age and sex related variations in serum apoA1 and apoB must have to be considered. In the present study serum apoA1 levels showed minor variations with age in normal males and females. In males apoA1 levels slightly declined after the age of 44 yrs. But in females a relative increase in serum apoA1 levels were observed after the age of 44 yrs and then the levels declined after the age of 54 yrs. Sex related differences in serum apoA1 levels were clearly significant. In every age group females were having significantly raised serum apoA1 levels than males. Even in the older age group significant difference in serum apoA1 levels were observed between normal males and females. Similar age and sex related variations were also observed in serum HDL cholesterol levels. ApoA1 is a constituent apolipoprotein of HDL and any change in apoA1 levels may affect the cholesterol uptake property of HDL. ApoA1 plays a key role in reverse cholesterol transport. It also activates LCAT which is essential for cholesterol esterification ₁₅. Relatively raised serum apoA1 levels in females could be responsible for the raised HDL cholesterol levels in them.

On the contrary serum apoB levels continuously increased with age in both normal males and females. No decline in its levels was observed in either sex. In every age group, males were having relatively raised serum apoB levels than females. A continuous increase in serum apoB levels in both the sexes may indicate a physiological increase in risk of CAD with advancing age .This risk seemed to be more pronounced in males having increased number of atherogenic particles (apoB) and decreased number of anti-atherogenic particles (apoA1). Very similar results were reported in Swedish population study. ₇ It was reported that apoB levels increased with age in both the sexes while serum apoA1 showed minor age related variations. The same study reported similar sex related differences in apoA1 and apoB levels as observed in the present study. Age groups taken in the Swedish study were different from the present study but that did not affect the results. Another study in Mexican population also reported similar results ₁₆. Although the results of the Swedish and Mexican studies were similar to the results of the present study, slight variations in the average levels of these apolipoproteins were observed. In the Mexican population study, relatively lower mean levels of serum apoA1 and apoB were reported as compared to the results of the present study. This strengthens the need to obtain more data regarding these apolipoproteins in population-based studies worldwide. LDL cholesterol levels also undergo similar age and sex related variations in normal males and females as were observed in serum apoB levels. A positive coefficient of correlation was observed between serum LDL cholesterol levels and apoB levels. ApoB plays an important role in lipid metabolism. It maintains the integrity of LDL and also facilitates delivery of cholesterol to the peripheral cells. Any variation in apoB levels could alter the cholesterol content in LDL leading on to the observed variations.

CAD Patients (males and females) had significantly low serum apoA1 and HDL cholesterol levels and significantly raised serum apoB and LDL cholesterol levels as compared to age and sex matched normal individuals (males and females). In every age group serum apoB/apoA1 ratio was significantly higher in CAD patients compared to normal individuals indicating increased atherogenicity. Raised apoB/A1 ratio has been associated with increased risk of CAD ₁₇. Significant difference in serum apoB levels of CAD patients and normal individuals has already been reported in our previous published data ₁₈. Low serum apoA1 levels have been reported in patients suffering from coronary artery disease₁₉. This clearly shows that apolipoprotein A1 and B are good candidates to act as risk factors for CAD. Similar results were also reported in one study in Indian population. ₂₀. Though this study reported that apoA1 and B could as better predictors in evaluating the risk of coronary artery disease, the absolute values of these apolipoproteins reported were significantly lower from the present study. This may be due to other factors such as difference in geographical distribution or different dietary habits, and cultural and behavioral factors of the population studied which may affect the levels of these apolipoproteins. Moreover the study design was different from that of the present study.

Conclusions

However this study concluded that serum apoB and apoA1 levels differ significantly with age and sex of a person and could act as good candidates in evaluating the risk of CAD. The number of cases in each age group may not be an indicative of CAD prevalence due to small sample size. This study emphasizes the need to have more data regarding the distribution of these apolipoproteins within different geographic regions and in other populations especially in developing countries due to high disease burden. Data regarding age and sex related variations in apoA1 and apoB in larger population could help to establish their reference levels in our country.

Study limitations

Acknowledgement