Original Article

Lipoprotein lipase activity is decreased in a large cohort of patients with coronary artery disease and is associated with changes in lipids and lipoproteins

Correspondence to: John J. P. Kastelein

	ABSTRACT

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Lipoprotein lipase (LPL) is crucial in the hydrolysis of triglycerides (TG) in TG-rich lipoproteins in the formation of HDL particles. As both these lipoproteins play an important role in the pathogenesis of atherosclerotic vascular disease, we sought to assess the relationship between post-heparin LPL (PH-LPL) activity and lipids and lipoproteins in a large, well-defined cohort of Dutch males with coronary artery disease (CAD). These subjects were drawn from the REGRESS study, totaled 730 in number and were evaluated against 75 healthy, normolipidemic male controls. Fasting mean PH-LPL activity in the CAD subjects was 108 (46) mU/ml, compared to 138 (44) mU/ml in controls (P < 0.0001). When these patients were divided into activity quartiles, those in the lowest versus the highest quartile had higher levels of TG (P < 0.001), VLDLc and VLDL-TG (P = 0.001). Conversely, levels of TC, LDL, and HDLc were lower in these patients (P = 0.001, P = 0.02, and P = 0.001, respectively). Also, in this cohort PH-LPL relationships with lipids and lipoproteins were not altered by apoE genotypes. The frequency of common mutations in the LPL gene associated with partial LPL deficiency (N291S and D9N carriers) in the lowest quartile for LPL activity was more than double the frequency in the highest quartile (12.0% vs. 5.0%; P = 0.006). By contrast, the frequency of the S447X LPL variant rose from 11.5% in the lowest to 18.3% (P = 0.006) in the highest quartile.

This study, in a large cohort of CAD patients, has shown that PH-LPL activity is decreased (22%; P = 0.001) when compared to controls; that the D9N and N291S, and S447X LPL variants are genetic determinants, respectively, in CAD patients of low and high LPL PH-LPL activities; and that PH-LPL activity is strongly associated with changes in lipids and lipoproteins.—Henderson, H. E., J. J. P. Kastelein, A. H. Zwinderman, E. Gagne, J. W. Jukema, P. W. A. Reymer, B. E. Groenemeyer, K. I. Lie, A. V. G. Bruschke, M. R. Hayden, and H. Jansen. Lipoprotein lipase activity is decreased in a large cohort of patients with coronary artery disease and is associated with changes in lipids and lipoproteins. J. Lipid Res. 1999. 40: 735–743.

Supplementary key words: REGRESS study, VLDL, LDL, HDL, cholesterol, triglyceride

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The role of lipoprotein lipase (LPL) in the modulation and determination of plasma lipid and lipoprotein levels is now well recognized. This is particularly evident in individuals who have a genetic deficiency of LPL (Type 1 hyperlipoproteinemia) and present with markedly elevated chylomicron levels and decreased VLDL, LDL, and HDLc concentrations. Individuals heterozygous for these null genetic defects, with approximately 50% activity, often also show abnormal lipid and lipoprotein levels in fasting plasma (1) (2) (3) (4) (5). Variations of LPL activity in normal subjects are likewise known to influence plasma lipids. This is particularly evident for HDLc, where studies in healthy adults (6) (7) (8) (9), twins (10), and dyslipidemic patients (11) (12) (13) have shown a statistically significant positive correlation with fasting post-heparin plasma LPL (PH-LPL) levels. This finding is in keeping with the hydrolytic action of LPL on triglyceride in the triglyceride-rich lipoproteins and with the entry of the excess surface phospholipid, cholesterol, and apolipoproteins into the HDL pool. The relationship of PH-LPL to fasting plasma TG levels is less clear, as the findings of a negative correlation in healthy adults (6) (14) (15) (16), dyslipidemic, obese and diabetic subjects (16) (17) (18) (19), has not been substantiated in other studies (20) (21) (22) (23) (24).

Significant difficulties in the assessment of some of these studies have been the small numbers of subjects and the resulting low discriminatory power, the presence of mixed genders, and the measurement of LPL activity in different laboratories, with differing assay methods. Here we have re-examined this question in a large single-gender cohort, comprising 730 Dutch males with angiographically defined coronary artery disease. Further design features of significance and relevance were the assay of PH-LPL levels in one laboratory using a single assay procedure and a rigorously controlled protocol of heparin injection and blood withdrawal for LPL determination.

Here we demonstrate that PH-LPL levels are lower in CAD subjects when compared to normolipidemic controls with a 22% decrease in the mean activity level. We further demonstrate unequivocally that PH-LPL activity in male CAD patients correlates inversely with fasting plasma TG and directly with HDLc levels. These correlations are in keeping with the role of LPL in the lipolysis of lipoprotein triglyceride and the generation of HDL particles. Also, the apoE genotype in these subjects was found to be of minor significance in determining the relationships between PH-LPL levels, with lipids and lipoproteins.

We, and others, have previously shown that the D9N and N291S variants in the LPL gene are associated both in vitro and in vivo, with lowered catalytic activity (16) (25) (26) (27) (28). We, therefore, determined the frequency of these variants in our CAD cohort and demonstrate that carrier status for these variants is a genetic determinant of low PH-LPL in CAD patients. The joint frequency of these variants in patients in the lowest quartile of PH-LPL levels was more than double that of the frequency in subjects in the highest quartile. Conversely, we demonstrate that the S447X gene variant is a likely contributory factor to the determination of PH-LPL levels in those subjects in the upper quartile of LPL activities.

Collectively these data from the REGRESS study suggest that depressed LPL catalytic function is a significant risk factor for CAD.

	MATERIALS AND METHODS

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Patients
Post-heparin plasma LPL activity measurements were available from 730 of the 885 subjects in the REGRESS study (29) which was designed as a double-blind, placebo-controlled, multi-center study to assess the effect of pravastatin treatment on the progression and regression of coronary atherosclerosis. No selection bias was evident for the 730 individuals examined. All patients were males of Caucasian descent, below 70 years of age, and had angiographically documented coronary artery disease (>50% stenosis of a major vessel). Patients were specifically excluded who had unstable angina or who suffered a myocardial infarction within the preceding 6 months. All patients had to have total cholesterol levels between 4 and 8 mmol/L and triglyceride levels below 4 mmol/L.

Controls (n = 75) were drawn from the general population, and were healthy, normolipidemic males, off lipid-lowering medication, and between 25 and 55 years of age with a mean age of 46 (5.1) years. All patients participating in the REGRESS trial signed written informed consent and the study protocol was approved by all institutional review boards of all respective hospitals.

Lipid and lipoprotein analysis
All laboratory tests were carried out at the Lipid Reference Laboratory, Academic Center, Amsterdam. Serum cholesterol, HDL cholesterol, and triglycerides were measured on fasting blood samples by standard methodologies (29). LDL cholesterol was calculated according to the Friedewald formula (30). The Lipid Reference Laboratory is an international member of the USA National Cholesterol Reference Method Laboratory Network chaired by the Centers for Disease Control and Prevention (Atlanta, GA).

DNA analysis
In a search for LPL mutations associated with lowered catalytic activity, DNA from all patients in the lowest quartile of LPL activity was subjected to mutation analysis by PCR and density gradient gel electrophoresis (DGGE), with sequencing, where indicated. This search covered the promoter region (-450 to -36) and the translated sequence of exons 1–10 of the LPL gene. The PCR primers used for the amplification of individual exons are given in Table 1. In addition, the entire subject cohort was screened for the Asp9Asn (D9N), Asn291Ser (N291S), and Ser447Stop (S447X) mutations in the LPL gene. This was achieved by PCR and restriction endonuclease digestion of amplified product, as described previously (16) (31) (32).

ApoE genotypes were determined by PCR and digestion with the restriction endonuclease Hha1 as described (33).

Post-heparin plasma lipase activity
Lipase activities were obtained on enrollment and were determined in fasting plasma, separated from blood collected after IV heparin (50 IU/kg). Patients were kept sedentary for 20 min after which heparin was injected as a bolus in normal saline. Twenty min later blood was drawn from the alternate arm and immediately placed on wet ice for transport to the central laboratory. All samples were received within 6 h of collection. LPL was determined after inhibition of hepatic lipase (HL) by co-incubation with a goat antibody to human HL (14). All activity assays utilized gum acacia-stabilized [³H]trioleoylglycerol as substrate. Released fatty acids were extracted as calcium salts and counted in a liquid scintillation counter.

The number of subjects in this cohort was large enough to allow for subdivision into groupings of varying LPL levels, which increased the comparative value of the study. Subjects were therefore partitioned into quartiles of PH-LPL activities. Following current convention, quartiles 2 and 3 were combined to give categories of subjects with low, intermediate, and high levels of PH-LPL.

Statistical analysis
The distribution of PH-LPL activity was checked for its shape and was transformed by the Box-Cox method for stabilization and normalization. The association between PH-LPL activity and baseline patient characteristics was assessed using ANOVA, univariate and multiple regression analysis. The frequency of mutations in different quartiles of LPL activity was tested using a two-sided Fishers Exact test.

	RESULTS

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Patient characteristics
PH-LPL levels were determined in 730 patients. The mean age of this cohort was 56 ± 8.1 years; whereas 73% were current users of ß-blockers and 87% reported a past history of smoking. In the group as a whole ( Table 2), no correlation was evident between PH-LPL activity, age (r = 0.05; P = 0.11), BMI (r = 0.01; P = 0.22), and systolic or diastolic blood pressure (r = 0.04; P = 0.16 and r = 0.03; P = 0.97, respectively). Other characteristics, notably CAD risk factors, CAD-related parameters, or pharmacological treatment, did not differ among patients in the lowest, intermediate, or highest quartiles of LPL activity (Table 2).

LPL-activity and lipid levels
Mean PH-LPL activity for the whole group was 108 mU/mL (SD = 46) with values ranging from 13 to 293 mU/mL and having a median of 103 mU/mL. The distribution of LPL activity was skewed to the right (Kolmogorov-Smirnov test: P = 0.002). The maximum likelihood estimate of the power parameter of the Box-Cox transformation was 0.4 with a 95% confidence interval (CI) of 0.32–0.56. Consequently, we used the square-root transformation (power = 0.5) to normalize and standardize the LPL distribution. In addition, mean and median of the square-root distribution of LPL activity were almost equal (10.16 vs. 10.15) and the Kolmorov-Smirnov test showed a P value of 0.58, indicating Gaussian distribution. The square root of LPL activity was subsequently used in all statistical inferences. The percentage variance of TG and HDLc attributable to PH-LPL is equal to the squares of their respective correlations (reported in Table 4), and were found to be (-0.02)² = 4% and (0.025)² = 6.3%, respectively.

Last, we divided the 730 patients into quartiles of LPL activity. The first quartile (PH-LPL, 13–77 mU/mL) contained 191 patients. The second (78–103 mU/ml) and third quartiles (104–132 mU/mL) contained 179 and 181 patients, respectively, and the fourth quartile (133–293 mU/mL) contained 179 patients (Table 2). The sample size in the first quartile is not equal to that of the others as the boundary between the first and second quartiles bisected a large number of patients who yielded PH-LPL activities of exactly 77 mU/ml. All these patients were therefore included in the first quartile.

Comparison of PH-LPL levels in REGRESS subjects and controls
PH-LPL activities were available for 75 normolipidemic controls, with a mean level of 138 (44) mU/mL ( Table 3). CAD patients and controls had significantly different LPL activities, 108 (46) versus 138 (44) mU/mL; P < 0.0001, which indicates a decrease of 22% in CAD patients. Significance was still obtained (P = 0.001) if the study subjects were divided into those on or off beta-blockers, 106 (42) (n = 543) and 114 (53) mU/ml (n = 188), respectively.

Lipids and lipoproteins in different quartiles of LPL activity
Patients in the lowest versus the highest quartile of LPL activity had higher levels of triglycerides {log transformed; 0.58 (0.43) vs. 0.35 (0.42) mmol/l, P < 0.0001} and higher levels of both VLDL-cholesterol and VLDL-triglycerides, and IDL-triglycerides {log transformed; -0.69 (0.84) vs. -1.26 (1.08) mmol/l, P = 0.002; -0.09 (0.83) vs. -0.62 (0.93), P = 0.002, respectively; -2.18 (0.83) vs. -2.49 (0.60), P = 0.02} ( Table 4). Conversely, levels of total cholesterol {5.96 (0.85) vs. 6.15 (0.84) mmol/l, P = 0.01}, LDL-cholesterol {4.21 (0.80) vs. 4.43 (0.77) mmol/l, P = 0.026} and HDL-cholesterol {0.86 (0.26) vs. 1.02 (0.23) mmol/l, P < 0.0001} were significantly lower in patients in the lowest versus the highest quartile of LPL activity. IDL-cholesterol {log transformed; -1.92 (0.84) vs. -2.08 (0.86) mmol/l, P = 0.42} levels, were not statistically different (Table 4).

PH-LPL activity correlated positively with LDL-cholesterol (r = 0.14; P = 0.011) and HDL-cholesterol (r = 0.25; P < 0.0001), but just missed significance (r = 0.13; P = 0.059) for total cholesterol. Conversely, a significant but negative association was evident for PH-LPL activity and triglycerides (r = -0.20; P < 0.0001). Values for triglycerides, VLDL, and IDL-triglycerides, were based on log-transformation, with means ± SD being given in the tables. Plots of PH-LPL activity versus plasma triglyceride and HDL-cholesterol are depicted in Figure 1A and 1B, respectively.

View larger version (17K): [in this window] [in a new window]   Figure 1. (A) A plot of post-heparin plasma lipoprotein lipase (PH-LPL) activity versus plasma TGs and (B) versus high density lipoprotein cholesterol (HDLc) for the 730 subjects from the REGRESS study. Solid lines represent the estimated regression lines between PH-LPL activity and TG and HDLc, respectively. Dotted lines indicate the 95% confidence interval around these regression lines.

ApoE genotypes
ApoE genotypes were available for 606 of the 730 subjects in this study cohort; no bias was discernable in the ascertainment of these genotypes. Statistically significant associations between PH-LPL activities, and lipid and lipoprotein levels were evident in each genotype grouping, with significance in the E3/3 cohort (n = 356) only being very similar to that of the associations in the total subject cohort. Here, significance was shown for PH-LPL and plasma TG, TC, LDLc, and HDLc. Weaker associations were found in the other genotype divisions with the combined E2/2 + E2/3 subjects failing to show significance between PH-LPL activities and either TG or HDLc levels, but reaching significance for total and LDL cholesterol. The combined E4/3 + E4/4 subjects only gave significant associations between PH-LPL, and TG and HDLc.

Contribution of LPL gene mutations to lowered PH-LPL activity in REGRESS subjects
Functional variants of LPL have been shown to occur relatively frequently in the general population (34). Therefore, to determine the contribution of mutations in the LPL gene to decreased PH-LPL activity, DNA samples of the 191 CAD patients in the lowest quartile of activity (77 mU/mL) were subjected to comprehensive mutation analysis of the promoter and exonic regions of the gene.

As anticipated, sequence changes were detected in exons 2, 6, and 7, which, respectively, represent the Asp9Asn (D9N), Asn291Ser (N291S), and Ser447'Stop (S447X) amino acid substitutions, previously described (27) (35) (36). The -93T/G promoter mutation (37) was found in this group, and in each case was linked with the D9N mutation. No additional base substitutions were identified in these subjects, other than the three known (35) (38) silent base substitutions in exons 3 (G₄₀₅'A), 4 (G₄₃₅'A), and 8 (C₁₁₆₄'A), which occurred at carrier frequencies of 7.5, 3.3, and 1.5%, respectively. Individuals in the other quartiles were not subjected to detailed mutation analysis but were screened for the D9N, N291S, and S447X mutations. The distribution and frequency of carriers of these LPL variants is given in Table 2. In the entire cohort we found 27 D9N and 37 N291S carriers. One of these patients carried both amino acid substitutions. Carriers of the S447X allele totaled 118; 4 were homozygous, while 6 also carried either a D9N or a N291S substitution. The total number of compound heterozygotes detected was 7, and these individuals were excluded from the analysis. No carriers of the predominant Caucasian mutation, Gly188Glu (G188E) were detected in this cohort.

Frequencies for D9N, N291S, and S447X carriers differed significantly between the lowest and highest quartiles of LPL activity. The combined frequency of D9N and N291S carriers in the lowest quartile was more than double the frequency in the highest quartile (12.0% vs. 5.0%: P = 0.006), while conversely in the high quartile the S447X frequency rose to 18.3% (11.5% vs. 18.3%: P = 0.006) as depicted in Table 5. These 447stop frequency differentials are similar to those reported previously by us for a smaller subgroup of the REGRESS cohort (32).

	DISCUSSION

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The determination of PH-LPL levels in subjects with CAD, from the REGRESS study, provided a unique opportunity for a rigorous appraisal of PH-LPL levels, and the relationships of these levels with circulating lipids and lipoproteins, particularly as a large number of single gender subjects were available together with a highly controlled laboratory assay for PH-LPL activity.

To our knowledge, our study is the first to demonstrate directly through lipase activity measurements, that males with CAD have lower mean levels of PH-LPL than normal controls. Earlier studies (39) (40) (41) have examined this aspect but have not been able to record differing mean LPL activities; however, the numbers of subjects in the cohorts examined, in each case, were low. A large number of our study subjects were on beta-blockers, which have been suggested to inhibit LPL activity (42) (43). However, even when we excluded these individuals, a significant reduction in mean PH-LPL activity was obtained for the CAD patients. Another potential factor of influence to be considered here would be that of advancing age where a decrease in PH-LPL activity has been reported (44). However, the difference in the mean ages between the control and CAD subjects was not a factor in our study as neither of these cohorts yielded a significant correlation of PH-LPL activity with age.

The finding of lower PH-LPL activities in our study cohort would suggest that this group is enriched in individuals who have reduced PH-LPL levels and would thereby implicate a reduction in PH-LPL activity as an etiological factor in CAD. This would most likely be on the basis of the atherogenicity of the small dense LDL, low HDLc, elevated plasma TGs, and prolonged post-prandial lipemia accompanying depressed PH-LPL activities (1) (45) (46). This conclusion is supported by several studies that showed that men with hypoalphalipoproteinemia and mild hypertriglyceridemia have significantly enhanced postprandial lipemia (46) (47) (48). Also, a preponderance of small, LDL particles has been linked to increased risk of myocardial infarction, and a dense and protein-rich LDL has proved to be characteristic of patients with CAD (49) (50) (51).

Plasma total triglyceride was found to correlate negatively with PH-LPL activity in our study cohort (Figure 1a). This finding is similar to that of previous studies (6) (14) (15) (17) and confirms this relationship in the largest cohort of CAD patients examined to date. This relationship was manifest by both the VLDL and IDL fractions. HDLc was positively correlated with PH-LPL activity, again confirming and extending previous findings (6) (7) (8) (10) (11) to a large cohort of CAD patients. These relationships in our subjects are in accordance with the role of LPL in the metabolism of the triglyceride-rich lipoproteins, and the maintenance of HDL levels. LPL is located on the capillary endothelium and is active in the lipolysis and removal of the triglycerides in chylomicrons, VLDL, and IDL. A reduction in LPL activity would therefore result in an increase in plasma TG, through slower TG hydrolysis, and a decrease in HDL due to decline in the rate of surface component influx into the HDL pool.

Apolipoprotein E is an important component of the clearance pathways for triglyceride-rich lipoproteins, through its participation in their uptake through a receptor-mediated process. Three isoforms are common in humans (E2,3,4) and are known to independently influence lipid and lipoprotein levels (51). They are therefore an important factor to consider in the evaluation of the impact of other components of the same clearance pathway on lipid and lipoprotein levels. In this context, a recent study in 52 healthy males (13) provides evidence that apoE may modulate the effect of variations of LPL activity on lipid and lipoprotein levels. However, unexpectedly, this effect was limited to carriers of the E2 isoform (E2/2 and E2/3; n = 12), which showed statistically significant correlations between PH-LPL, and VLDL and HDL levels. No correlations reached significance in the other two divisions, E3/3 (n = 29) and E3/4, E4/4 (n = 11). We have therefore also studied the impact of apoE isoforms on PH-LPL relationships in our cohort of males and found no specific effect of the E2 isoform ( Table 6). In fact, the association between PH-LPL and TGs was weakest in the E2/2 + E2/3 grouping; this is in direct contrast to the previous study (13). All three genotype categories showed statistically significant relationships, with the E3/3 division in particular, paralleling the associations of the total subject cohort, by yielding highly significant (P = <0.001) relationships with plasma–TG, LDLc, and HDLc. These data contradict prior findings and, originating from a much larger cohort, suggest that the apoE polymorphism is of minor importance in assessing the relationships between PH-LPL activity and plasma lipid and lipoprotein levels.

We have shown here that CAD subjects in the REGRESS study have a lower PH-LPL level than normal subjects. In an attempt to characterize the factors determining the lower level in these subjects, we determined their carrier frequency for LPL variants known to be associated with lower catalytic activities. Only two previously reported functional variants, D9N and N291S, were detected in this cohort, clearly indicating that no other additional mutations in the LPL gene are likely to contribute to lowered LPL activity levels in the Dutch population. The contribution of these variants to the depression of PH-LPL levels was evident in comparisons of their carrier frequencies in the lowest versus the highest quartile divisions of the REGRESS cohort. Both variants were detected at an increased prevalence in the former division, where a joint carrier frequency of 12.0% was found. This is more than double that of the 5.0% found in the upper quartile division. Clearly, while these variants contribute significantly to the etiology of the lower PH-LPL levels in this division, other factors must be involved. The identity of these factors is undetermined as likely candidates such as age, BMI, and beta-blockers did not differ significantly among the quartile divisions.

Several studies examining the relationships between LPL gene variants and lipid and lipoprotein levels in humans (16) (32) (52) (53) (54) (55) (56) (57) have provided direct and indirect evidence that the S447X variant is associated with an increase in LPL catalytic activity which manifests with lower plasma TGs and higher HDLc levels in carriers of this variant. Based on these data, the frequency of carrier status for the S447X variant was predicted to be elevated in subjects in the upper quartile of PH-LPL activities. This prediction was validated as the carrier frequency of 18.3% (n = 179) was significantly elevated (P = 0.006) above the 11.5% recorded for individuals (n = 191) in the lowest quartile of PH-LPL activities. These data thus significantly strengthen the in vivo evidence that the S447X variant manifests with a higher catalytic activity when compared to wild type LPL. In vitro data supporting this hypothesis have been sought in several mutagenesis and transfection systems (26) (58) (59) (60) but thus far, little difference has been discovered between the functional activities of this variant and wild type LPL which may point to a selective mechanism for the in vivo effect.

In conclusion, this study demonstrates that post-heparin plasma LPL activity is an important determinant of lipid and lipoprotein levels in humans, relatively independent of secondary factors such as age and BMI. In addition, the REGRESS study provides evidence that low PH-LPL activity is a risk factor for CAD and that common mutations in the LPL gene can contribute to this risk through their impact on the variation of PH-LPL levels in these patients.

	ACKNOWLEDGMENTS

The REGRESS study was supported by Bristol-Myers Squibb Co, Princeton, NJ. This work was also supported by grants from the universities of the respective authors and the Medical Research Councils of Holland, Canada, and South Africa. We are grateful to all the cardiologists and co-workers who made plasma and DNA samples from patients available to us.

Manuscript received July 20, 1998; and in revised form December 1, 1998.

Abbreviations: LPL, lipoprotein lipase; TG, triglycerides; PH, post-heparin; CAD, coronary artery disease; HDL, high density lipoproteins; LDL, low density lipoproteins; DGGE, density gradient gel electrophoresis; PCR, polymerase chain reaction

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