INTRODUCTION

High density lipoproteins (HDL) are macromolecular, pseudo-micellar complexes whose most well-known function is to transport cholesterol from peripheral tissues to the liver for metabolism and excretion, a process known as reverse cholesterol transport (RCT). These lipoproteins also have antithrombotic, anti-inflammatory, vasodilatory, immunosuppressive and antioxidant properties ¹. This has led to the consideration for years of HDL as protective molecules for cardiovascular disease (CVD), which is considered a global public health problem and the first cause of death worldwide; it caused 17.8 million deaths in 2017 ². In the population of Maracaibo, high prevalence of low levels of high-density lipoprotein cholesterol (HDL-C) has been previously observed ³; however, the coronary risk seems to be lower than in other populations with lower frequency of this type of lipid alteration ⁴.

Although there is epidemiological evidence that HDL-C is an independent predictor of CVD, clinical pharmacological trials focused on its quantitative improvement have not demonstrated benefit in reducing cardiovascular events or mortality ⁵. This paradoxical phenomenon has been justified due to the heterogeneity and complexity shown by these molecules, whose functionality is the fundamental characteristic in their role as cardiovascular “protector” ⁶. In this regard, there seems to be a close relationship between the function and the subfractions or subspecies of HDL-C ⁷.

De Lalla et al. ⁸ described for the first time the distribution of HDL-C subfractions and identified two subclasses: HDL2, the least dense (1.063-1.125 g/mL) and richer in lipids; and HDL3, the most dense (1.125-1.21 g/mL) and relatively rich in proteins. HDL2 and HDL3 seem to have different functions. HDL3 has the ability to receive free cholesterol from the endothelium or macrophages, through the ATP-binding cassette transporter A-1 (ABCA- 1), increasing its size with the decrease of its density, thus transforming into HDL2, which is responsible for the RCT by binding to SR-B1 receptors. Therefore, the role of each subfraction in cardiometabolic health could be different ⁹.

Epidemiologic findings related to subfractions are diverse and controversial, that is why no international diagnostic and management guidelines for dyslipidemia currently recommend routine evaluation in patients with cardiovascular risk. However, several reports show the potential role that these subfractions would have as markers for HDL functionality, and the modulating effect that drugs would have on CVD ^{7 , 10}.

In Latin America, especially in Venezuela, although low HDL-C is a frequent dyslipidemia, there are few or no reports describing the behavior of HDL subfractions or their relationship to other cardiovascular risk factors. Therefore, the aim of this study was to perform a clinical-epidemiological analysis of HDL2 and HDL3 subfractions in an adult population in the city of Maracaibo, Venezuela.

MATERIALS AND METHODS

RESULTS

Characteristics of the population

Table 1 shows general characteristics of the studied sample, made up of 359 individuals, of which 51.5% (n = 185) were female. The average age of the population was 39.4 ± 15.2 years. Regarding HDL-C behavior according to sex (Figure 1), it was shown that concentration of this lipoprotein was significantly higher in women than men (47.2 ± 13.4 mg/dL vs. 40.1 ± 9.4 mg/dL; p < 0.001). Also, statistically significant differences were found in the concentration of HDL3 (women: 28.9 ± 11.9 mg/dL vs. men: 25.1 ± 8.6 mg/dL; p = 0.004) and HDL2 (women: 18.2 ± 9.2 mg/dL vs. men: 15.0 ± 8.3 mg/dL; p = 0.001).

Figure 1 Epidemiological behavior of HDL-C concentration and subfractions according to sex. 

HDL-C subfraction percentiles

Table 2 shows the percentile distribution of HDL-C subfractions by sex. The average HDL3 in women was 26.7 mg/dL (P₂₅-P₇₅: 19.9-36.9); 17.6 mg/dL (P₂₅-P₇₅: 10.7-24.6) for HDL2; and 0.66 (P₂₅-P₇₅: 0.35-1.02) for the HDL2/HDL3 ratio. While the HDL3 average in men was 24.8 mg/dL (P₂₅-P₇₅: 18.9-30.5); 13.8 mg/dL (P₂₅-P₇₅: 9.2-18.1) for HDL2; and 0.56 (P₂₅-P₇₅: 0.35-0.83) for the HDL2/HDL3 ratio.

HDL-C subfractions according to socio-demographic characteristics and habits

Table 3 shows the behavior of HDL2 and HDL3 in individuals with low HDL-C according to sociodemographic variables. Women from socioeconomic strata IV-V presented significantly lower values of HDL3 compared to those from higher socioeconomic strata (p < 0.001). Likewise, women from strata IV-V 4presented a higher HDL2/HDL3 ratio with respect to those from strata I-II (0.9 ± 0.5 vs. 0.7 ± 0.6, respectively; p = 0.012).

Regearding race, a higher concentration of HDL2 and a higher HDL2/HDL3 ratio were evidenced in American Indians compared to white-Hispanic individuals (HDL2: 17.8 ± 5.0 mg/dL vs. 9.8 ± 4.2 mg/dL; p = 0.040 and HDL2/HDL3: 1.1 ± 0.45 vs. 0.5 ± 0.3; p = 0.007, respectively). On the contrary, in men with low HDL-C, no significant differences were found in the subfraction averages with respect to the sociodemographic variables.

As for psychobiological habits, non-smoker women presented higher levels of HDL3 with respect to ex-smokers (22.4 ± 6.6 mg/dL vs. 17.1 ± 7.6 mg/dL, respectively, p = 0.034). On the contrary, men with low HDL-C only showed significant differences between the level of leisure physical activity and the HDL2/HDL3 ratio (p = 0.038), without observing important differences between the ratio values for each activity type.

HDL-C subfractions according to clinical-metabolic characteristics

Regarding clinical-metabolic alterations, it was observed (Table 4) that women with low HDL-C and hypertriglyceridemia showed significantly lower HDL3 and HDL2 serum concentrations compared to those with normal triglycerides (TG) (p = 0.033 and p = 0.034, respectively). Similar findings have been reported in women with elevated us-CRP, in whom lower HDL3 concentration and HDL2/HDL3 ratio were observed, compared to those with normal us-CRP (p < 0.001 and p = 0.011, respectively). In men with hypertension a significantly lower concentration of HDL2 (p = 0.031), HDL3 (p = 0.028) and ratio HDL2/HDL3 (p = 0.028) was evidenced. Likewise, these individuals showed a significantly lower concentration of HDL2 in the presence of IR (p = 0.050) and MS (p = 0.003) and men with elevated us-CRP also showed a lower concentration of HDL3 (p = 0.011).

Table 4 HDL Subfractions according to clinical-metabolic characteristics and sex of subjects with low HDL-C 

TG: triacylglycerides; BMI: body mass index; AC: abdominal circumference; IR: insulin resistance; us-CRP: ultra-sensitive C-reactive protein; MS: metabolic syndrome.

† Criteria according to IDF/NHLBI/AHA consensus; ^¶ Criteria according to EPSMM (HOMA2-IR≥2).

* t-Student test to compare between two categories, or single-factor ANOVA test to compare between three or more categories in patients with normal HDL; no significant differences between group means were observed.

DISCUSSION

HDL are molecules that can exist in multiple isoforms, and heterogeneity is one of their main properties. Although the HDL-C concentration has been inversely correlated to the risk of CVD and atherosclerosis, heterogeneity confers additional effects to its anti-atherogenic properties, which are attributed to the various molecules are part of the HDL-C ⁶.

For decades it has been suggested that HDL subfractions could be more directly related to the occurrence of cardiovascular events; however, despite positive results, there is no conclusive evidence about which subclass is the most important in this context ^{10 , 18 , 19}. This probably influences the absence of international recommendations for using these biochemical parameters. In fact, HDL subfractions are not mentioned in the most recent North American or European guidelines for diagnosis and management of dyslipidemia.

Given this controversial scenario and the lack of reports evaluating HDL subclasses in Latin American populations, especially in Venezuela, this study describes the main clinical and epidemiological characteristics of HDL subfractions in the population of the city of Maracaibo, the second most important city in Venezuela in terms of population and economy. In fact, during the sample collection period, Maracaibo presented a high frequency of cardiovascular risk factors and deleterious psychobiological habits, such as sedentarism and high consumption of saturated fats ^{20 , 21}.

It is important to show average values and dispersion measures for any epidemiological analysis of a laboratory measurement with scarce reports from a region or country. When comparing the HDL-C subfraction results from this study, with other populations, we found different figures. For example, in a European population, Kim et al. ²² found a higher HDL3 average (women: 49 ± 11 mg/dL and men: 39 ± 11 mg/dL), and the HDL2 average was higher in women (women: 14.7±7.8 mg/dL and men: 8.5±5.3 mg/dL). In a study by Koumaré et al. ²³, the HDL3 average in Burkina Faso was the same for men and women (26.2 mg/dL), while the HDL2 average in men was 15.4 mg/dL, and 18.9 mg/dL in women, similar values to those obtained by this research.

It is important to mention that there are differences in the averages of HDL subfractions in subjects with low HDL-C. In relation to the sociodemographic data, American Indians presented higher levels of HDL2 and HDL2/HDL3 compared to white-Hispanic individuals; this data differs from the study carried out by Martin et al. ²⁴, where there was no evident relation between ethnic groups and the different subfractions of HDL-C.

As for the clinical-metabolic characteristics, it has been described that dyslipidemias represent a frequent cardiovascular risk factor and that high levels of triglycerides frequently coexist with low HDL-C and alterations in the subfraction distribution ²⁵. Our findings show that the levels of both subfractions were significantly lower in women with hypertriglyceridemia, which differs from the results of Jia et al. ²⁶, who observed that hyperlipidemic subjects presented lower levels of HDL2 but higher levels of HDL3 when compared with normolipidemic subjects. Similarly, Gou et al. ²⁷ found that HDL2 levels were significantly lower in women with hypertriglyceridemia than in those with normal triglyceride levels, while the HDL3 average was higher in the latter group. Such differences could be explained by the method used for obtaining HDL-C subfractions and by the size of the sample; however, these results suggest an inverse relationship between hypertriglyceridemia and HDL2.

Also, low HDL-C levels have been associated with low-grade inflammation, IR, and visceral obesity. In this regard, the results obtained show decreased HDL3 levels in subjects with elevated us-CRP in both sexes, while those with MS and IR showed lower HDL2 levels in men. These results match with the findings of the ELSA-Brazil study, one of the few Latin American reports that shows characteristics of HDL-C subclasses in our region, in which the concentration of HDL-C and its subfractions were inversely associated with low grade inflammation, IR and MS ²⁷. Similarly, another Brazilian study that evaluated the impact of IR on the metabolism of different lipoproteins showed lower percentages of the largest subfractions (HDL2 and HDL3) in subjects with IR ²⁸, these findings evidence the role that the structure and function of HDL-C would have as cardioprotective lipoproteins.

The molecular mechanisms involved in the inverse relationship between HDL-C subfractions and inflammatory mediators, such as us-CRP, have not yet been clarified. However, the lower HDL3 levels in subjects with elevated us-CRP could be interpreted as a smaller number of nascent lipoproteins available to mobilize and externalize cholesterol from endothelial foam cells, which would lead to the activation and accumulation of the triggers of the inflammatory phenomenon. Another Latin American study shows lower levels of HDL2 and HDL3 specifically in Peruvian diabetic subjects ²⁹, an association that was not significant in our study.

The values observed in this study for the HDL2/HDL3 ratio only showed relation with the socioeconomic status, race, and us-CRP in women; while in men, relations were observed with physical activity, blood pressure and MS. In this regard, Moriyama et al. ³⁰ carried out several investigations in Japanese population and showed that changes in the HDL2/HDL3 ratio correlate inversely with abdominal circumference and IR, and positively with healthy lifestyle habits, which is why it is used as a useful marker for MS and atherogenic conditions in that population.

Regarding the limitations to this study, the sample size does not allow the generalization of the results to the entire population; the cross-sectional design makes it impossible to establish relationships of causality or recommendation of clinical use of HDL-C subfractions, and the socioeconomic changes that affect the study population during recent years could influence the results shown.

In conclusion, HDL-C subfractions have variable clinical-epidemiological behavior in adult individuals of Maracaibo’s population, lower averages in men, differences in socioeconomic levels only in those with low HDL-C, and no predominance of any subclass according to sociodemographic, clinical and biochemical characteristics. Therefore, it is suggested to deepen the study of these lipoproteins in the Latin American population, emphasizing their relationship with different disorders that lead to a higher cardiometabolic risk.