Paraoxonase (PON1) is associated with familial combined hyperlipidemia

Atherosclerosis 199 (2008) 87–94

Paraoxonase (PON1) is associated with familial combined hyperlipidemia Thomas M. van Himbergen a,b,∗ , Lambertus J.H. van Tits b , Ewoud ter Avest b , Mark Roest a , Hieronymus A.M. Voorbij a , Jacqueline de Graaf b , Anton F.H. Stalenhoef b a

Department of Clinical Chemistry and Haematology, UMC Utrecht, Utrecht, The Netherlands b Department of Medicine, Division of General Internal Medicine, Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands

Received 10 August 2007; received in revised form 1 October 2007; accepted 22 October 2007 Available online 21 December 2007

Abstract Familial combined hyperlipidemia (FCH) is a common genetic lipid disorder of which the molecular basis still remains to be elucidated. Since the HDL-associated enzyme serum paraoxonase (PON1) is associated with variation in serum lipids and lipoproteins, we determined whether variation in PON1 also contributes to the FCH phenotype. The study population consisted of 32 well-defined families with FCH, including 103 FCH patients and 240 normolipidemic relatives (NLR). In addition to plasma lipids and lipoproteins we determined PON1 activity (arylesterase- and paraoxonase activity) as well as the common genetic variants −107C > T, 55L > M and 192Q > R in the PON1 gene. The arylesterase activity was significantly higher in FCH patients when compared to NLR (P < 0.001). In the total population, the PON1 genetic variants associated with the highest arylesterase activity (−107CC and 55LL) also associated with higher levels of total cholesterol, apolipoprotein B, triglycerides and VLDL-cholesterol and decreased levels of HDL-cholesterol. In support, the combination of the −107CC with the 55LL genotype associated with a significant increased risk for FCH when compared to the −107TT/55MM genotype (odds ratio 5.0 (95% CI, 1.3–19.1, P = 0.02)). In conclusion, in this population of subjects from well-defined families with FCH, PON1 is biochemically and genetically associated with FCH. © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Epidemiology; Enzymes; Genetics; Lipid disorders

1. Introduction Familial combined hyperlipidemia (FCH) is a common genetic lipid disorder. Affected subjects characteristically show elevation of plasma total cholesterol, triglycerides and apolipoprotein (apo) B, and are more prone to develop premature cardiovascular disease (CVD) [1]. The prevalence of ∗ Corresponding author at: Lipid Metabolism Laboratory, Jean Mayer United States, Department of Agriculture Human Nutrition Research Center on Aging at Tufts University, 711 Washington Street, Boston, MA 02111, USA. E-mail address: [email protected] (T.M. van Himbergen).

0021-9150/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2007.10.017

FCH in the general population has been reported to be up to 5.7% [2], and FCH has been estimated to cause approximately 10% of premature CVD [3]. The pathophysiological mechanism underlying FCH is believed to be a hepatic overproduction of apoB-100 containing lipoprotein particles, i.e. VLDL and LDL, resulting in increased plasma cholesterol, triglycerides and apoB levels [4–6]. In addition, lower concentrations of HDL cholesterol and increased amounts of small dense LDL (sdLDL) particles can be observed in patients with FCH [7–11]. Despite more than 30 years of research, the genetic origin of FCH remains obscure [12]. The heterogeneous lipid profile, however, indicates that FCH is a multifactorial disease in which several genes affect the lipid and lipoprotein metabolism.

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We hypothesized that the HDL-associated enzyme paraoxonase (PON1) contributes to the FCH phenotype. A growing body of evidence shows that PON1 is an important enzyme in the metabolism of serum lipids and lipoproteins and therefore PON1 may also be of importance in the expression of lipid disorders: many epidemiological studies have shown that PON1 associates with HDL-cholesterol levels [13–18], in addition, total cholesterol, triglycerides, LDLcholesterol, VLDL-cholesterol and apoB levels have also been shown to associate with PON1 [18–22]. Although recent investigations have given much insight into the functioning of PON1, the exact mechanisms underlying these associations of PON1 with the lipids and lipoproteins remain to be elucidated. Furthermore, the role of PON1 in diseases characterized by lipoprotein disorders has remained largely unexplored, and until today no studies have addressed the effects of PON1 on the FCH phenotype. Although PON1 can hydrolyze a wide range of substrates, recent studies have demonstrated that PON1 is in fact a lactonase, catalyzing both the hydrolysis and the formation of a number of lactones [23–25]. To study the role of PON1 in humans, serum PON1 activity and concentration can be determined by monitoring the hydrolytic activity towards paraoxon (paraoxonase activity) and phenyl acetate (arylesterase activity), respectively [26,27]. Although the same active site of the PON1 enzyme is responsible for the hydrolysis of the two substrates, the paraoxonase activity is greatly affected by a single polymorphism, a glutamine (Q)to-arginine (R) transition at position 192 (192Q > R) in the PON1 gene [28–30]. The arylesterase activity on the other hand, can be used to estimate PON1 serum concentrations [27]. Moreover, the arylesterase activity is mediated by the same His115 -His134 dyad which is responsible for PON1’s native lactonase activity [31]. Genetically, the 55L > M polymorphism in the coding region and the −107C > T polymorphism in the promoter region of the PON1 gene have the strongest impact on PON1 concentrations in blood [32,33]. We investigated whether the arylesterase and paraoxonase activity of the PON1 enzyme, as well as the PON1 genetic variants 192Q > R, 55L > M and −107C > T are associated with the FCH phenotype. For this we studied the effects of PON1 in a population of 32 well-defined FCH families consisting of 103 patients with FCH and 240 normolipidemic relatives (NLR). Additionally, to gain further insight into the role of PON1 in the lipid metabolism, we also investigated the association between PON1 and lipids and lipoproteins in the total study population as well as in the subgroups of the NLR and the patients with FCH.

were diagnosed with FCH [34]. This diagnosis was based on our recently published nomogram [34]. Briefly, plasma triglycerides and total cholesterol levels, adjusted for age and gender, and absolute apoB levels were included in a nomogram to calculate the likelihood of having FCH. A subject was defined to be affected by FCH when the probability was above 60%, provided that the diagnostic phenotype was also present in at least one first-degree relative and premature CVD (i.e. before the age of 60) was present in at least one individual in the family. After withdrawal of lipid-lowering medication for 4 weeks, blood was drawn after an overnight fast. The ethics committee of the Radboud University Nijmegen Medical Centre approved the study protocol and all subjects gave informed consent. 2.2. Laboratory measurements Plasma total cholesterol and triglycerides were determined by enzymatic, commercially available reagents (BoehringerMannheim, Germany, catalog No. 237574 and Sera Pak, Miles, Belgium, catalog No. 6639, respectively). LDLcholesterol was calculated according to the Friedewald formula. VLDL-cholesterol was determined by ultracentrifugation [35], HDL-cholesterol was determined by the polyethylene glycol 6000 method [36], and total plasma apoB concentrations were determined by immunonephelometry [37]. Serum PON1 enzyme activity was measured by paraoxonase and arylesterase activity according to methods previously described [26,27]. The inter-run variation coefficients for the laboratory measurements were as follows: total cholesterol (1.7%), triglycerides (1.9%), LDL-cholesterol (2.3%), VLDL-cholesterol (4.3%), HDL-cholesterol (1.5%), apoB (6.0%), paraoxonase activity (2.5%) and arylesterase activity (6.2%). The PON1 55L > M and 192Q > R genetic variants were determined by restriction fragment length polymorphism (PCR-RFLP) using primers and restriction enzymes as described by Humbert et al. [30]. The −107C > T polymorphism was detected by amplifying the DNA region of interest using PCR primers described by Leviev and James [38], followed by genotype determination according to the previously described iFLASH method [39]. The following infrareddye (IRD) labeled allele-specific oligonucleotides were used: 5 -IRD700-GGAGGGGCGGAGCG-3 for detection of the −107C allele and 5 -IRD800-GGAGGGGTGGGGCG-3 for detection of the −107T allele. PCR products were successfully obtained in 329, 333 and 324 samples for the 55L > M, 192Q > R and −107C > T, respectively.

2. Methods

2.3. Statistical analyses

2.1. Study population

Baseline variables were tested for normal distribution. Comparisons among the FCH patients and the NLR were performed using the Independent Samples t-test for normally distributed continuous variables and the χ2 test for categorical

The study population consists of 343 subjects, from 32 well-defined FCH families. One hundred and three subjects

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variables. Triglycerides and VLDL-cholesterol showed skewed distribution, baseline comparisons were made with the Mann–Whitney test for non-normally distributed continuous variables and correlations were based on log-transformed data. In order to correct for the lack of genetic independence of the subjects (i.e. the family structure), the family number was entered into the regression models as a covariate. Linear regression analysis was used to evaluate the differences in arylesterase- and paraoxonase activities and plasma lipid and lipoprotein concentrations among the genetic variants of the 192Q > R and −107C > T polymorphisms. Age, gender, family structure and −107C > T (CC versus CT/TT), 55L > M (LL versus LM/MM) or 192Q > R (QQ versus QR/RR) were entered into the model as explanatory variables and, alternately, arylesterase activity, paraoxonase activity, total cholesterol, apoB, log-transformed triglycerides, HDLcholesterol and log-transformed VLDL-cholesterol were entered into the model as dependent variables. Odds ratios for developing FCH among PON1 −107C > T and 55L > M genotype combinations were calculated using logistic regression. The homozygote −107TT in combination with 55MM served as reference category for the other genotype combinations. The FCH phenotype served as outcome variable. Odds ratios were controlled for age, gender and family structure. Correlation coefficients for PON1 arylesterase- and paraoxonase activity with plasma lipid and lipoprotein parameters were calculated using the SPSS partial correlation procedure controlling for age, gender and family structure. In case of a missing value, the correlation pair was excluded. The association between arylesterase and VLDLcholesterol in the patients with FCH was investigated using a linear regression model with VLDL-cholesterol, age, gender, family structure and the −107C > T and 55L > M genotypes as explanatory variable and arylesterase activity as dependent variable. A P-value smaller than 0.05 was considered statistically significant and all calculations were performed with SPSS 11.5.

3. Results The characteristics of the 103 patients with FCH and the 240 NLR are described in Table 1. There was no difference in gender distributions among FCH patients and the NLR, but the FCH group was significantly older (P < 0.001). Characteristically, plasma total cholesterol, triglycerides, apoB, LDL- and VLDL-cholesterol concentrations were significantly higher in the FCH group when compared to the NLR (P < 0.001, for all relations). Interestingly, although HDLcholesterol levels were lower, the PON1 arylesterase activity was higher in individuals affected with FCH (P < 0.001). Paraoxonase activity was also higher in patients with FCH compared to the NLR, but this difference was not signifi-

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Table 1 Baseline characteristics of the normolipidemic relatives (NLR) and patients with familial combined hyperlipidemia (FCH)

Gender (male/female) Age (years) Total cholesterol (mmol/L) Triglycerides (mmol/L) HDL-cholesterol (mmol/L) LDL-cholesterol (mmol/L) VLDL-cholesterol (mmol/L) ApoB (mg/L) Arylesterase activity (U/mL) Paraoxonase activity (U/L)

NLR (n = 240)

FCH (n = 103)

117/123 44 ± 15 5.2 ± 1.0 1.2 (0.9–1.6) 1.3 ± 0.3 3.5 ± 0.9 0.4 (0.2–0.6) 960 ± 221 90.1 ± 19.2 253.4 ± 182.3

45/58 51 ± 15* 6.9 ± 1.2* 2.9 (2.0–4.1)* 1.1 ± 0.3* 4.2 ± 1.2* 1.1 (0.7–1.9)* 1372 ± 256* 101.3 ± 22.8* 289.4 ± 206.4

Values presented as mean ± standard deviation, except for gender, which is presented as the number of male and females, and triglycerides and VLDLcholesterol, which are presented as median (inter quartile range). * P < 0.001 for the comparison with the NLR group.

cant (Table 1). Furthermore, the PON1 specific activity (i.e. the paraoxonase activity per arylesterase activity unit) was not elevated in FCH when compared to the NLR (data not shown). The PON1 arylesterase- and paraoxonase activity were strongly influenced by the −107C > T, 55L > M and 192Q > R polymorphisms. The −107CC, 55LL and 192QR/RR genotypes were associated with the highest arylesterase and paraoxonase activity (Table 2). The −107CC variant was also associated with increased total cholesterol (P < 0.01), apoB (P < 0.05), triglycerides (P < 0.01) and VLDL-cholesterol (P < 0.01) levels. Additionally, the 55LL variant was associated with lower HDL-cholesterol levels (P < 0.01) and higher triglycerides (P < 0.05) and VLDL-cholesterol (P < 0.05) levels. The 192Q > R genotype, which is of strong influence on the paraoxonase activity, was not associated with any of the lipid or lipoprotein traits. Life-style determinants like smoking and the use of lipid lowering medication prior to the start of the study were not independent predictors of the PON1 activities (data not shown). When the PON1 genotypes were considered as separate risk factors for FCH, they did not reach statistical significance (data not shown). However, there was significant linkage between the −107C > T and the 55L > M genotypes (χ2 P < 0.001), and because the −107CC genotype was associated with higher total cholesterol, apoB, triglycerides and VLDL-cholesterol levels, and the 55LL genotype was associated with higher triglycerides and VLDL-cholesterol levels and lower HDL-cholesterol levels, we investigated whether the combined genotypes contributed to the risk on developing FCH. The low concentration variants 55MM and −107TT were combined and served as reference group. In the total study population, 63 subjects (20%) were carrier of the −107CC/55LL genotype combination which was associated with a five-fold higher chance to develop FCH when compared to the −107TT/55MM genotype (OR, 5.0 (95% CI, 1.3–19.1, P = 0.02)) (Fig. 1). Intermediate effects were observed for the other genotype combinations (Fig. 1). When considering the FCH patients and their NLR as separate

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Table 2 Arylesterase- and paraoxonase activities and plasma lipid and lipoprotein concentrations among PON1 −107C > T, 55L > M and 192Q > R genotypes in total study population −107C > T

Number of subjects Arylesterase activity (U/mL) Paraoxonase activity (U/L) Total cholesterol (mmol/L) Triglycerides* (mmol/L) HDL-cholesterol (mmol/L) LDL-cholesterol (mmol/L) VLDL-cholesterol* (mmol/L) ApoB (mg/L)

55L > M

CC

CT/TT

LL

LM/MM

89 110.2 ± 18.4† 339.3 ± 211.2† 6.0 ± 1.5‡ 1.54 (1.09–2.73)‡ 1.24 ± 0.33 3.8 ± 1.3 0.62 (0.35–1.12)‡ 1127 ± 331§

235 87.0 ± 18.5 237.3 ± 175.7 5.6 ± 1.2 1.39 (0.98–2.13) 1.27 ± 0.32 3.7 ± 0.9 0.46 (0.26–0.80) 1063 ± 291

141 100.2 ± 19.5† 353.2 ± 212.6† 5.8 ± 1.4 1.54 (1.09–2.45)§ 1.22 ± 0.28‡ 3.7 ± 1.1 0.53 (0.31–0.96)§ 1098 ± 316

188 88.0 ± 19.9 195.5 ± 137.7 5.7 ± 1.2 1.35 (0.97–2.10) 1.30 ± 0.34 3.7 ± 1.0 0.46 (0.26–0.80) 1072 ± 290

192Q > R

Number of subjects Arylesterase activity (U/mL) Paraoxonase activity (U/L) Total cholesterol (mmol/L) Triglycerides* (mmol/L) HDL-cholesterol (mmol/L) LDL-cholesterol (mmol/L) VLDL-cholesterol* (mmol/L) ApoB (mg/L)

QQ

QR/RR

187 91.1 ± 19.6§ 127.3 ± 44.2† 5.7 ± 1.3 1.42 (1.0–2.36) 1.27 ± 0.34 3.7 ± 1.0 0.47 (0.28–0.96) 1074 ± 295

146 95.8 ± 21.5 439.1 ± 158.7 5.7 ± 1.3 1.44 (1.06–2.21) 1.26 ± 0.29 3.8 ± 1.0 0.47 (0.27–0.76) 1099 ± 308

Values presented as mean ± standard deviation, except for triglycerides and VLDL-cholesterol which are presented as median (inter quartile range). * Between group comparisons are based on log-transformed data. P-values are based on linear regression and adjusted for age, gender and family structure. † P < 0.001. ‡ P < 0.01. § P < 0.05 when compared to carriers of CT/TT, LM/MM or QR/RR.

groups, the −107CC/55LL genotype was present in 26 subjects (27.1%) and 37 subjects (16.7%), respectively. In order to understand the basis of elevated PON1 arylesterase- and paraoxonase activities in FCH, we investigated the correlations of the PON1 activities with lipid

and lipoprotein parameters in the FCH patients and their unaffected relatives (Table 3). First, in the NLR group there was a positive correlation between arylesterase activity and HDL-cholesterol (P < 0.01), but also total cholesterol, LDLcholesterol and apoB correlated positively with arylesterase activity (P < 0.001 for all relations). Within the FCH patients, arylesterase activity was not associated with HDLcholesterol nor with apoB levels, but rather with the total

Table 3 Correlation coefficients for PON1 arylesterase and paraoxonase activity with plasma lipid and lipoprotein concentrations in the normolipidemic relatives (NLR) and patients with familial combined hyperlipidemia (FCH)

Fig. 1. The probability to develop FCH among combinations of the PON1 −107C > T and 55L > M genotypes. Genotypes were grouped according to arylesterase activity. The homozygote −107CC in combination with 55LL showed the highest arylesterase activity of 110.8 U/mL (±18.0 U/mL standard deviation) and the homozygote −107TT in combination with 55MM the lowest activity of 73.8 (±11.6 U/mL standard deviation). The remaining genotypes were grouped as intermediate genotypes and showed an intermediate arylesterase activity of 90.3 (±19.0 U/mL standard deviation). The −107TT/55MM genotype combination served as a reference category. The number of genotypes per category is shown in parentheses. ORs were adjusted for age, gender and family structure. * OR, 5.0 (95% CI, 1.3–19.1, P = 0.02).

Total cholesterol ApoB HDL-cholesterol LDL-cholesterol log VLDL-cholesterol log triglycerides

NLR (n = 240)

FCH (n = 103)

AE activity

POX activity

AE activity

POX activity

0.334* 0.275* 0.213† 0.277* 0.049 0.099

0.098 0.141‡ 0.008 0.096 0.041 0.056

0.276† 0.088 0.066 −0.048 0.240‡ 0.237‡

0.097 0.017 0.068 −0.046 0.074 0.047

Correlations were controlled for age, gender and family structure. AE, arylesterase and POX, paraoxonase. * P < 0.001. † P < 0.01. ‡ P < 0.05.

T.M. van Himbergen et al. / Atherosclerosis 199 (2008) 87–94 Table 4 Multiple regression models for variables associated with arylesterase activity in patients with FCH Standardized beta coefficients

P value

Model 1* Age (years) Gender (M/F) Family structure log VLDL (mmol/L)

−0.312 0.242 −0.031 0.325

0.003 0.023 0.751 0.005

Model 2† Age (years) Gender (M/F) Family structure log VLDL (mmol/L) −107C > T (CC/CT/TT) 55L > M (LL/LM/MM)

−0.202 0.126 0.017 0.087 −0.553 −0.141

0.019 0.152 0.835 0.367 <0.001 0.121

Arylesterase activity (U/mL) served as outcome variable. * Variance explained 9%. † Variance explained 45%.

cholesterol, triglycerides and VLDL-cholesterol concentrations (P < 0.01, for all relations). The observed correlations between paraoxonase activity and the lipid and lipoprotein parameters were due to the strong association of paraoxonase activity with arylesterase activity which reflects the serum PON1 concentration. The strength of the correlation between paraoxonase activity and lipid parameters declined after adjusting for arylesterase activity (data not shown). As we have shown, the relationship of PON1 with the lipid profile is different in FCH patients and their NLR. In concordance, there are also differences in the variance in arylesterase activity attributable to the −107C > T and 55L > M polymorphisms. Using a regression model adjusted for age, gender and family structure, we found that in patients with FCH 45% of the variation in arylesterase activity could be explained by the genetic variants at position −107 and 55, while in the NLR this was only 31% (data not shown). Interestingly, the association between arylesterase activity and VLDL-cholesterol in the FCH patients, was no longer significant after adjustment for the −107C > T and 55L > M polymorphisms, thus suggesting an intermediate effect for the PON1 genotypes (Table 4).

4. Discussion This is the first study to investigate the contribution of PON1 to the FCH phenotype. We observed that PON1 arylesterase activity was increased in FCH patients. There was also a genetic basis for this observation since the PON1 −107CC and 55LL genotypes, associated with an increased arylesterase activity, were also associated with a 5-fold increased risk on FCH. Finally, the examination of PON1 with the lipid profile in FCH patients and their unaffected relatives separately, revealed remarkable differences. In the NLR, PON1 specifically associated with higher levels of apoB, HDL- and LDL-cholesterol, in the FCH patients con-

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versely, PON1 specifically associated with the higher levels of VLDL-cholesterol and triglycerides. At present, the complex genetics of FCH is still not fully understood and the fact that FCH is a genetic heterogeneous disease complicates the identification of major contributing genes [12,40]. Many genes involved in the lipid metabolism have been demonstrated to associate with FCH [41], thus warranting the investigation of others. Previously, we found indications that PON1 plays a role in the metabolism of HDL-C in familial hypercholesterolemia patients [15], and accordingly, in the present study we show an association of PON1 with FCH. A limitation of our study, however, is that the genetic evidence is based on a relatively small number of cases which resulted in wide confidence intervals for the effect estimates. Nevertheless, the genetic association of PON1 is in line with the increased arylesterase activity observed in the FCH patients. These findings suggest a significant role for PON1 in the disturbed lipid metabolism associated with the FCH phenotype and therefore, further investigations on the genetics of FCH should include PON1 as a candidate gene. In general, PON1 is thought to attenuate the oxidation of LDL, based on in vitro findings that purified PON1 inhibits the accumulations of lipid peroxides in LDL [42], and therefore may have an antiatherogenic function in the human body. Apart from inhibition of LDL oxidation, there is evidence from animal and in vitro models that paraoxonase can protect the HDL particle from oxidation and preserve the integrity and function of HDL [43,44]. These findings are supported by epidemiological studies in different populations demonstrating that PON1 contributes to variation in plasma levels of HDL-cholesterol [15–18]. Thus it is hypothesized that the preservation of HDL by PON1 may beneficially influence the lipid metabolism and together with PON1’s antioxidative function, forms the basis for PON1’s antiatherogenic potential. However, our findings that increased PON1 activity associates with an increased risk for FCH is in sharp contrast with PON1’s assumed antiatherogenic functions and indicate that the exact mechanism by which PON1 functions in the lipid metabolism and the atherogenesis is still not clear and requires further investigation. In blood, PON1 is located exclusively on the HDL particle and, as expected, we found a positive correlation between PON1 and HDL-cholesterol in the NLR. In FCH, however, the association of PON1 with lipids and lipoproteins was different. Surprisingly, arylesterase activity was higher in FCH patients while HDL-cholesterol levels were decreased in these subjects. Apparently, the higher turnover or the reduced production of HDL-cholesterol in FCH patients does not influence PON1, suggesting that there is no interaction between PON1 and HDL in these patients. Rather a correlation of arylesterase with VLDL-cholesterol and triglycerides was present in FCH, and therefore, one could hypothesize that there is a physical displacement of the PON1 particle from the HDL to VLDL. In line with this hypothesis, a recent in vitro study demonstrated that VLDL not only promoted the

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secretion of PON1 from cells but also served as an accepting vector for PON1 in the absence of HDL [45]. We investigated whether in FCH the PON1 protein was present on the VLDL particle by fractionating the serum of five FCH patients using FPLC and measuring PON1 activity in each lipoprotein fraction. We found that the PON1 activity was present only in the HDL fraction, and we could not detect any PON1 activity in the VLDL fractions of these patients (unpublished results). This may implicate that after the initial acceptance by VLDL all the PON1 is quickly transferred to the HDL as seen in vitro [45]. Although HDL-cholesterol levels are lower in the FCH patients, HDL is still abundantly present to serve as a carrier for PON1. VLDL may enhance the secretion of PON1 into blood, but does not carry PON1 in vivo. Context-dependent transcriptional regulation of PON1 may underlie the differential associations of PON1 with lipids and lipoproteins observed in FCH patients and the NLR. Transcription factors, which play an important role in the regulation of the cholesterol homeostasis [46], were also found to regulate the expression of PON1 in vitro [47,48]. In the NLR, the PON1 genotypes only explain 31% of the variation in arylesterase activity while in FCH 45% of the variation could be explained by these genotypes. These data indicate that after PON1 is expressed, in the NLR it is more prone to influences by environmental factors than in the FCH patients. Furthermore, our data show that the association between VLDL and arylesterase activity diminishes after adjustment for the effects of PON1 genotypes. This indicates that PON1 genotype is an intermediate step in the association between VLDL and arylesterase activity and that PON1 and VLDL levels may be the result of regulation by the same mechanism. Our data indicate that PON1 plays a role in the disturbed lipoprotein metabolism in FCH patients. As mentioned earlier, we find differences in the association of PON1 with lipids and lipoproteins among FCH and NLR: arylesterase activity correlated with triglycerides and VLDL-cholesterol in the FCH subgroup and these correlations were not found in the NLR subgroup. Instead, in the NLR subgroup, arylesterase activity correlated with apoB, LDL- and HDL-cholesterol levels. The consistency of these findings with recent data presented by Rozek et al. is remarkable [22]. In a control population consisting of healthy men without severe coronary artery disease, they found that arylesterase activity correlated with HDL-cholesterol and ApoA1, whereas in men with severe carotid artery disease, a significant correlation was observed for arylesterase activity with total cholesterol, triglycerides and VLDL-cholesterol but not with HDLcholesterol [22]. Apparently, in both FCH and carotid artery disease, the association of PON1 with the lipid metabolism shifts from HDL towards the non-HDL particles. In addition to Rozek et al., we also found that PON1 genotypes associate with variation in lipids and lipoprotein. In general, in the human body the effect of a single genetic variant is the result of complex interactions with the environment. Rozek

et al. speculated that the differences in patients and controls might be the result of context-dependent transcription regulation of PON1 and lipoprotein components, but did not find the effects of PON1 genotype to support this hypothesis. In the present study we do find these effects and therefore our data confirm that the observed differences in association with lipoproteins may be the result of a common regulation in transcription. In summary, we show evidence that PON1 is biochemically and genetically associated with FCH. In addition, we find that PON1 associates differentially with lipids and lipoproteins in FCH and NLR. These data suggest that PON1 is involved in the lipoprotein metabolism. The mechanisms of such an involvement require further investigation in wellcontrolled experimental studies.

Acknowledgements This study was financially supported by the Netherlands Heart Foundation (Research Grant #2001.038). The authors thank Anneke Hijmans and Arjan Barendrecht for their technical assistance.

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