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Department of Medicine (J.d.G., G.M.v.d.V., E.t.A., A.F.H.S.), Division of General Internal Medicine, Radboud University Nijmegen Medical Centre, 6500 HB Nijmegen, The Netherlands; and Laboratory of Vascular Medicine (G.M.D.-T.), Erasmus Medical Center, 3000 CA Rotterdam, The Netherlands
Address all correspondence and requests for reprints to: Jacqueline de Graaf, M.D., Ph.D., Department of Medicine, Division of General Internal Medicine 463, Radboud University Nijmegen Medical Centre, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands. E-mail: J.degraaf{at}aig.umcn.nl.
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Abstract |
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Objective: The objective of the study was to investigate whether plasma RLP-C concentrations are elevated in patients with FCH and contribute to the increased prevalence of CVD.
Design, Setting, Participants: In this cross-sectional study, we studied RLP-C levels in 37 FCH families comprising 582 subjects, of whom 134 subjects were diagnosed FCH based on total cholesterol, triglyceride, and apolipoprotein-B levels. Plasma RLP-C concentrations were determined using an immune-separation technique.
Results: For both men and women, the mean plasma RLP-C concentration (mmol/liter) was 2-fold elevated in FCH patients [0.59 (0.54–0.66) and 0.40 (0.37–0.43), respectively] compared with both normolipidemic relatives [0.27 (0.26–0.29) in male and 0.22 (0.21–0.23) in female, all P < 0.000]; and spouses [0.27 (0.23–0.31) in male and 0.24 (0.21–0.27) in female, all P <0.000]. Plasma RLP-C levels above the 90th percentile predicted prevalent CVD, independently of nonlipid cardiovascular risk factors [odds ratio 2.18 (1.02–4.66)] and triglyceride levels [odds ratio 2.35 (1.15–4.83)]. However, in both FCH patients and controls, RLP-C did not provide additional information about prevalent CVD over and above non-high-density lipoprotein cholesterol levels.
Conclusions: Patients with FCH have 2-fold elevated plasma RLP-C levels, which add to the atherogenic lipid profile and contribute to the increased risk for CVD. However, for clinical practice, non-high-density lipoprotein cholesterol is the best predictor of prevalent CVD.
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Introduction |
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The classical concept of the pathogenesis of CVD, based on lipid and lipoproteins, has implicated elevated cholesterol, particularly LDL-C, as the central atherogenic lipoprotein class. However, accumulating data suggest that apo-B containing lipoproteins other than LDL, particularly in the setting of mild to moderate hypertriglyceridemia, confer additional atherogenic risk beyond that due to LDL-C levels alone. FCH is an example of a lipid disorder with only mildly elevated plasma LDL-C levels but characteristic high apo-B levels due to increased VLDL-cholesterol (VLDL-C) and VLDL-triglyceride (VLDL-TG) levels, insulin resistance, and associated abnormalities. Recently, in a large observational study, the calculated plasma non-HDL-C concentration (LDL-C + VLDL-C) was a stronger predictor of cardiovascular events than plasma cholesterol alone (5, 6). Improvement of CVD predictability upon inclusion of VLDL-C emphasizes the proatherogenic role of TG-rich lipoproteins (TRL). Smaller, partially lipolyzed TRL remnants [i.e. remnant-like particles cholesterol (RLP-C)] are considered to be more atherogenic than larger newly secreted TRL because they can more readily penetrate the endothelial lining of the arterial wall (7). Several lines of evidence have implicated RLP-C as playing an etiologic role in atherogenesis, as recently reviewed (8, 9). In fact, RLPs have been present in atherosclerotic lesions (10).
Recently, an immunoaffinity separation method was introduced for assaying cholesterol in RLPs as RLP-C (11). Plasma concentrations of RLP-C have been higher in patients with CVD (12) and diabetes mellitus (13). In elderly patients, RLP-C rather than LDL-C was strongly associated with CVD (14). Increased RLP-C levels are a significant predictor of myocardial infarction in patients with vasospastic angina (15) and CVD (16), and have been strongly associated with angiographically verified progressions of focal coronary atherosclerosis (14). Recently, a high plasma level of RLP-C in patients with the metabolic syndrome was reported (17). In the metabolic syndrome, elevated levels of RLP-C were a risk factor for CVD and endothelial dysfunction, a predictor of coronary events (18).
The aim of the present study was to determine whether increased plasma RLP-C levels contribute to the FCH phenotype and its increased risk of CVD.
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Subjects and Methods |
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The study population consisted of 37 families, comprising 644 subjects, of whom 158 subjects were diagnosed as FCH patients. The diagnosis of FCH was based on plasma levels of total cholesterol, TG, and apo-B using the nomogram, as reported recently (19).
The normolipidemic relatives (n = 390) and spouses (n = 89), also included in this study population, served as two independent reference groups because of the similar genetic background of the relatives with the FCH patients. There were seven subjects who had missing data concerning their diagnostic criteria because of missing data for apo-B levels. All subjects filled out a questionnaire about their previous medical history, especially cardiovascular status, medication use, and smoking habits, as previously reported (19, 20). When the clinical investigator suspected the presence of CVD, further details and confirmation of the diagnosis were sought from the participant’s general practitioner, plus, if considered necessary from any relevant hospital records. In our study population, 56 subjects were identified with CVD, including 26 with angina pectoris, 25 with previous myocardial infarction, 10 with peripheral vascular disease, seven with stroke, and 23 underwent vascular surgery. In total, 45% (n = 25) of these subjects were diagnosed with CVD based on the presence of two or more manifestations of CVD.
After withdrawal of lipid-lowering medication for 4 wk and an overnight fast, venous blood was drawn by venipuncture. Body mass index (BMI) was calculated as body weight (kilograms) divided by the square of height (meters). The maximum hip and waist circumferences at the umbilical level were measured in the late exhalation phase while standing. The waist to hip circumference ratio (WHR) was calculated. Systolic and diastolic blood pressure was assessed twice with an automated blood pressure device (Dinamap; Critikon, Tampa, FL) in the right arm in a sitting position after a 5-min rest period; mean blood pressures were used for analysis. The ethical committee of the Radboud University Nijmegen Medical Centre approved the study protocol, and the procedures followed were in accordance with institutional guidelines. All subjects gave informed consent.
Laboratory measurements
VLDL was isolated from whole plasma by ultracentrifugation at a density of 1.006 g/ml for 16 h at 36,000 rpm in a fixed angle rotor (TFT 45.6 rotor; Kontron, Poway, CA), in a Beckman L7–55 ultracentrifuge (Beckman Coulter, Inc., Fullerton, CA). The polyethylene glycol 6000 method determined HDL-C (21). Subtraction of VLDL-C and HDL-C from plasma total cholesterol calculated LDL-C. Enzymatic, commercially available reagents (Roche Molecular Biochemicals, Germany, catalog no. 237574 and Sera Pak, Miles, Belgium, catalog no. 6639, respectively) determined cholesterol and TGs.
As recently described in detail elsewhere (22), immunonephelometry determined total plasma apo-B concentrations. Single-spin density gradient ultracentrifugation separated LDL subfractions (23). A continuous variable K represents the LDL subfraction profile of each individual. A negative K value (K 
–0.1) reflected a more dense LDL subfraction profile, and a positive K value (K > –0.1) reflected a more buoyant profile (24).
Glucose concentrations were measured in duplicate using the oxidation method (Glucose Analyser2; Beckman Coulter, Inc.). Plasma insulin concentrations were determined using a double antibody method with an interassay variability of 10.3% (25). Insulin resistance was assessed by the homeostasis model assessment (HOMA). The HOMA was calculated from the fasting concentrations of insulin and glucose using the following formula: HOMA = fasting serum insulin (µU/ml) x fasting plasma glucose (mmol/liter)/22.5 (26).
RLP-C assay
The RLP fraction was prepared using an immune-separation technique described by Nakajima et al. (11) and Campos et al. (27). Briefly, 5 µl serum was added to 300 µl mixed immunoaffinity gel suspension containing monoclonal antihuman apoA-I (H-12) and antihuman apoB100 (JI-H) antibodies (Immunoresearch Laboratories, Takasaki, Japan). The reaction mixture was gently shaken for 120 min at room temperature. After the supernatant was left standing for 15 min, 200 µl was withdrawn for the assay of RLP-C. Cholesterol in the RLP fraction (coefficient of variation < 3%) was measured by an enzymatic assay on a Cobas Mira S auto analyzer (ABX Diagnostics, Montpellier, France). So, the RLP-C fraction reflects cholesterol in both the apoB48-containing remnants and the apoB100-apoE remnants. Because of technical errors, plasma RLP-C levels could not be determined in 62 subjects, including 24 FCH patients; therefore, RLP-C levels were available in 582 subjects, including 134 FCH patients, 387 normolipidemic relatives, and 61 spouses.
Statistical analysis
The descriptive statistics expressed as means with SD are represented separately for FCH patients, normolipidemic relatives, and spouses. Variables like plasma RLP-C concentration, TG levels, non-HDL-C levels, and the HOMA index, with a skewed distribution, were logarithmically transformed for analysis. Generalized estimating equations were used to test differences in characteristics between subjects with FCH, spouses, and normolipidemic relatives because of possible correlated values within families. Also, odds ratios (ORs) with corresponding confidence interval (CI) as an estimate of risk for FCH or CVD were calculated using generalized estimating equations. Correlations between plasma RLP-C and variables were analyzed using Pearson correlation coefficients. The multiple linear regression test was used to select the variables that contributed independently to plasma RLP-C. Probability values < 0.05 were considered statistically significant. All analyses were computed using the STATA 8.0 software (StataCorp, College Station, TX).
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Results |
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Descriptive statistics for anthropometric measurements and biochemical variables in patients with FCH, normolipidemic relatives, and spouses are presented in Table 1
. The mean age of the group of subjects with FCH was significantly higher compared with the normolipidemic relatives and significantly lower compared with the spouses. As expected, the group of patients with FCH had a higher prevalence of CVD compared with both normolipidemic relatives and spouses. The mean BMI and WHR of FCH patients were significantly higher compared with the normolipidemic relatives. Systolic and diastolic blood pressure was significantly higher in patients with FCH compared with their normolipidemic relatives but did not differ from the spouses. Patients with FCH were more insulin resistant than the normolipidemic relatives and spouses as assessed by the elevated HOMA index. By definition, FCH patients were characterized by increased plasma total cholesterol, TG, and apo-B levels compared with normolipidemic relatives and spouses. Furthermore, FCH patients had a more atherogenic lipid and lipoprotein profile, as reflected by significantly lower plasma HDL-C levels, higher plasma LDL-C, and non-HDL-C levels, and the presence of small dense LDL, as reflected by a significantly lower K-value. In addition, significantly increased levels of VLDL-C and VLDL-TG were present in FCH compared with both normolipidemic relatives and spouses. The differences between the spouses and normolipidemic relatives are indicated in Table 1.
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The mean plasma RLP-C concentration in the FCH patients was significantly higher compared with both the normolipidemic relatives and spouses, indicating a 2-fold elevation of RLP-C in FCH patients (Table 2). The mean RLP-C levels in the normolipidemic relatives did not differ from the spouses. Plasma RLP-C levels differed between males and females (i.e. men had higher plasma RLP-C levels than women), which did only not reach statistical significance in the group of spouses. After adjustment for gender and age, the mean difference in plasma RLP-C concentration between FCH patients vs. spouses and normolipidemic relatives remained significant [0.46 (SD 0.38) vs. 0.25 (SD 0.45) and 0.23 (SD 0.36), respectively].
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). Characteristics of subjects with plasma RLP-C levels above the 90th percentile
In the non-FCH control group, plasma RLP-C levels above the 90th percentile were associated with older age, an increased BMI and WHR, and elevated blood pressure (Table 3). Furthermore, control subjects with high plasma RLP-C levels above the 90th percentile showed a more atherogenic lipid and lipoprotein profile characterized by higher total cholesterol and TG levels, lower HDL-C levels, higher apo-B levels, and non-HDL-C levels and more small dense LDL (i.e. lower K-values). Although plasma glucose levels were significantly higher in the control subjects with high RLP-C levels above the 90th percentile, no difference in HOMA index was found compared with the group with RLP-C levels < 90th percentile. Similar associations were found in the FCH group (data not shown).
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Plasma RLP-C levels in relation to lipid, lipoprotein, and insulin resistance parameters
Plasma RLP-C level showed significant univariate correlations in the control group with age, BMI, WHR, both systolic and diastolic blood pressure, total cholesterol, TGs, VLDL-C, VLDL-TG, HDL-C, non-HDL-C, LDL-C, apo-B, K-value, insulin, glucose, and the HOMA index (Table 4).
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Multiple linear regression analyses in the non-FCH control group and in the group of FCH patients showed that the variation in plasma RLP-C concentration could be enlightened for approximately 66 and 77%, respectively, by the variables gender and lipid and lipoprotein levels, including TGs, VLDL-C, non-HDL-C, LDL-C, and small dense LDL.
In both controls and FCH patients, gender, TG levels, and non-HDL-C levels were the main contributors in the explanation of the variation in RLP-C levels (62% in the non-FCH control group and 54% in the FCH-group).
Plasma RLP-C levels and CVD
In univariate analysis, a plasma RLP-C concentration above the standardized 90th percentile was associated with an increased risk for CVD in the non-FCH control group [OR = 2.38 (0.87–6.55)] and in FCH subjects [OR = 2.14 (0.75–6.12)], reaching statistical significance when the 2 groups were combined [OR 3.55 (1.97–6.39)] (Table 5, model 1). When putting this in a model together with nonlipid risk factors, including age, gender, smoking habits, HOMA, BMI, WHR, and blood pressure, plasma RLP-C concentration stayed an independent contributor to the prediction of CVD with an OR of 2.18 (1.02–4.66) for the total group. Both plasma levels of TGs and non-HDL-C were also predictors of prevalent CVD in univariate analysis in both FCH and the control group, as indicated in Table 5 (model 2 for TGs and model 3 for non-HDL-C). The independence of RLP-C as a predictor of CVD when including other lipid variables strongly correlated with RLP-C, was explored in model A and B, as presented in Table 5. When putting TG levels and RLP-C levels together in one model to predict prevalent CVD (model A, Table 5), the relative predictive power of RLP-C levels for CVD was higher in the FCH group, whereas plasma TGs contributed more to the prediction of CVD in the control group. For the total group, both plasma RLP-C and TG levels contributed independently to the risk of CVD. Adding non-HDL-C to the model (model B, Table 5) showed that non-HDL-C had the best predictive power for CVD in both the FCH and control groups.
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Discussion |
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Previous studies have shown that lipoprotein metabolism in FCH patients is disturbed, leading to remnant accumulation in the circulation, using different methodologies to isolate lipoprotein remnants (28, 29). Only one other study has reported plasma RLP-C levels in FCH (29). In this small study, 12 male FCH patients had a 6-fold increased plasma RLP-C concentration [1.54 ± 1.84 vs. 0.24 ± 0.05 mmol/liter (n = 14) in controls], whereas the plasma RLP-C concentration was only 2-fold higher in female FCH patients (n = 11) compared with controls (n = 13) (0.44 ± 0.42 vs. 0.17 ± 0.04 mmol/liter). This is most likely the result of higher TG levels compared with our population (TG 5.4 ± 5.2 vs. TG 2.79 ± 1.6 mmol/liter).
We show that men have higher RLP-C levels compared with women in both FCH patients and controls. In addition, plasma RLP-C levels increased with age in both males and females, confirming earlier reports in other populations (13, 30).
In our non-FCH control group, elevated plasma RLP-C levels are associated with obesity, insulin resistance, and an atherogenic lipid and lipoprotein profile characterized by elevated levels of plasma total cholesterol, TGs, apo-B, decreased levels of plasma HDL-C, and more small dense LDL. Similar associations have been reported previously (31, 32). We show that the major determinants of fasting RLP-C were gender, plasma TG level, and non-HDL-C, accounting for 72% of the variance in fasting RLP-C levels.
Strikingly, in the FCH group, no significant correlation of RLP-C levels with apo-B levels and even a negative correlation with LDL-cholesterol was observed. This is most likely explained by the fact that RLPs are highly heterogenous in size and composition; in normolipidemic subjects, RLPs are enriched in cholesterol, and its plasma concentration was correlated with plasma LDL-C concentration. However, when plasma TG levels increase, as in FCH, then the elution profile of RLP is shifted toward a larger-size particle, similar to that of a larger VLDL subfraction (VLDL1). So, the TG-rich component of RLP-C is responsible for the increased RLP-C concentration associated with hypertriglyceridemia because plasma TGs are then the major determinant of the size and composition of RLPs. Indeed the major determinants of plasma RLP-C levels among FCH patients were plasma TG levels.
The importance of plasma TG levels as an independent risk factor for CVD was recognized after the publication of a metaanalysis by Austin et al. (33), showing that plasma TG levels predict relative risk for CVD mortality in relatives of FCH patients. Epidemiological data from the Framingham study (34) already showed that plasma TG is an important risk indicator of CVD in women. Yarnell et al. (35) obtained additional evidence in a 10-yr follow-up study and confirmed several other studies (36). It is important to realize that in these studies (34, 35, 36), the role of TG as an independent risk predictor of CVD was investigated without considering all other atherogenic lipoproteins (i.e. small dense LDL). However, TGs are not significantly and independently associated with CVD in most prospective studies (37). The difficulty with using plasma or serum TG measurements to assess CVD risk may stem from the variability within the subspecies of TRL particles as well as the inverse association with HDL-C. It is not possible to distinguish TRLs that are atherogenic from those that are nonatherogenic simply by measuring plasma TG levels. Therefore, in the present study we use a specific assay to measure atherogenic RLP because plasma RLP-C levels have been associated with CVD and its risk factors. First, we show that increased plasma RLP-C levels are associated with an increased risk for CVD in non-FCH subjects and FCH patients, independently of nonlipid cardiovascular risk factors, including age, gender, smoking, obesity, insulin resistance, and blood pressure. Even when plasma TG levels are considered, we show that in the total group, RLP-C levels conferred additional risk of CVD; subjects with RLP-C levels above the 90th percentile have a 2.4-times increased risk of prevalent CVD, independent of plasma TG levels. Indeed, several clinical studies showed that plasma RLP-C offered independent assessment for CVD risk in addition to TG (38, 39). Furthermore, we show that in patients with FCH, the plasma RLP-C level is a better predictor of CVD than plasma TGs; these results support the concept of identifying more atherogenic remnant particles by measuring RLP-C instead of TG levels. However, most importantly for clinical practice, RLP-C did not confer an additional risk of prevalent CVD when non-HDL-C levels were considered; the best independent predictor of prevalent CVD in FCH patients was non-HDL-C.
In the control group, the plasma TG level is a better predictor for prevalent CVD than the RLP-C level. This could be related to the particle heterogeneity in RLP, which could affect the ability of RLP-C concentration to predict atherosclerotic risk. Therefore, in controls with low plasma TG levels, plasma RLP-C may not have the same clinical significance as they do in patients with FCH with hypertriglyceridemia. Indeed, in the Honolulu Heart Study (40), including normotriglyceridemic healthy men, RLP-C levels did not provide additional information about risk of CVD over and above TG levels, whereas the association between RLP-C and CVD was significant for the group with elevated TG levels. Further studies should examine whether difference in RLP composition may reflect a different risk on CVD in different populations. Thus, RLP-C may increase our knowledge on the identification of potentially more atherogenic lipoproteins. However, also in the group of nonaffected control subjects, RLP-C did not provide additional information for CVD risk prediction; for clinical practice, non-HDL-C is the best predictor of prevalent CVD.
However, it remains to be studied in large epidemiological studies to what extend the measurement of plasma RLP-C provides additional knowledge for the treatment goals of the individual patients.
Conclusions
Patients with FCH have 2-fold elevated plasma RLP-C levels. RLP-C levels predict prevalent CVD independent of nonlipid cardiovascular risk factors and independently of plasma TG levels, supporting the concept of identifying more atherogenic remnant particles by measuring RLP-C instead of TG levels. However, for clinical practice non-HDL-C is the best predictor of prevalent CVD in both FCH and nonaffected FCH control subjects.
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Acknowledgments |
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Footnotes |
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Disclosure Statement: All authors have nothing to declare.
First Published Online January 16, 2007
Abbreviations: apo, Apolipoprotein; BMI, body mass index; C, cholesterol; CI, confidence interval; CVD, cardiovascular disease; FCH, familial combined hyperlipidemia; HDL, high-density lipoprotein; HOMA, homeostasis model assessment; LDL, low-density lipoprotein; OR, odds ratio; RLP, remnant-like particles; TG, triglyceride; TRL, triglyceride-rich lipoprotein; VLDL, very low-density lipoprotein; WHR, waist to hip ratio.
Received September 7, 2006.
Accepted January 5, 2007.
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