OBJECTIVES:Paraoxonase (PON1) is a potent enzyme, physically associated with the high-density lipoprotein particle. PON1 may protect against cardiovascular disease (CVD), since it is capable of hydrolyzing oxidized LDL-cholesterol, thereby negating the detrimental effects of this lipoprotein on the arterial wall. DESIGN AND METHODS:In 187 patients with familial hypercholesterolemia, we studied the seven most common single nucleotide polymorphisms (SNPs) in both the coding and promoter sequences of PON1 (L55M, Q192R, T-107C, C-126G, G-162A, G-824A, and C-907G) in terms of PON1 activity and intima media thickness (IMT) of the carotid arterial wall, a validated surrogate marker for CVD. RESULTS:In concordance with several previous studies, we observed that the L55M, T-107C, G-162A, G-824A, and C-907G SNPs conferred PON1 activity towards phenylacetate, while this was not the case for the Q192R and C-126G SNPs. Importantly, in a multivariate regression analysis, G-824A proved to be an independent predictor of carotid IMT. Additionally, the two fully discordant homozygous haplotypes, C-907/G-824/G-162/C-126/T-107/55M versus -907G/-824A/-162A/-126G/-107C/L55, differed by 22% in carotid IMT (P = 0.007). CONCLUSIONS:Genetic variation at the PON1 locus has a strong influence on PON1 activity as well as on carotid IMT. These data indicate that PON1 is indeed involved in the pathogenesis of atherosclerosis. Whether this also translates into a role for PON1 in the occurrence of CVD events needs to be confirmed by large prospective studies in the general population.

Paraoxonase is an enzyme associated with the high-density lipoprotein (HDL) particle. It catalyses the hydrolysis of organophosphates and protects LDL from oxidative modification in vitro by hydrolyzing lipid peroxides, suggestive of a role for paraoxonase in the development of atherosclerosis. Two frequent mutations at the paraoxonase gene locus (PON1) underlie the leucine (Leu allele) → methionine (Met allele) and the glutamine(Gln allele) → arginine(Arg allele) aminoacid substitutions at residues 55 and 192, respectively. These polymorphisms have been associated with increased risk for cardiovascular disease (CVD) in several studies, while others have not found this association. Recently, another member of the PON gene family designated PON2 has been identified. While the PON2 gene product is expressed ubiquitously, its physiological role is unknown. A common polymorphism at codon 311 (Cys→Ser) in the PON2 gene has been described. In our study we assessed the frequency and genotype distribution of the PON1 and PON2 polymorphisms in 197 patients with familial hypercholesterolemia (FH), to determine the possible association between these mutations and susceptibility for CVD. The FH cohort group was divided into subjects with (n=83) and without (n=114) definite clinical manifestations of CVD (FH-Symptomatic and FH-Asymptomatic respectively). The control population consisted of 201 healthy normolipidemic blood donors. All subjects in this study were of Caucasian background. Genotypes were identified by PCR based analysis. With regard to the PON1 polymorphisms 55 and 192, no different distributions of allele frequencies were found between the groups studied. However, we did show an association between the PON2 311 polymorphism and CVD. The frequencies of PON2 Ser311 carriers (Ser/Ser and Cys/Ser) between FH-Symptomatic and both FH-Asymptomatic and controls did show a significant difference (P=0.01 and P=0.02 respectively). In the FH-Symptomatic population, surprisingly, no subjects were homozygous for PON2 Cys311, whereas in the FH-Asymptomatic population nine persons (7.9%) and in the control group 12 persons (6.0%) were homozygous. Our data indicate that the common PON2 polymorphism is associated with clinical manifestations of CVD in FH patients. While PON2 Ser311 carriers seem to be at risk, subjects with the Cys/Cys311 genotype are likely to be protected against the development of premature CVD.

Human serum paraoxonase (PON) is a high density lipoprotein (HDL) associated enzyme capable of hydrolyzing lipid peroxides in vitro. PON has recently attracted attention as a protective factor against oxidative modification of LDL and may therefore play an important role in the prevention of the atherosclerotic process. Two frequent mutations at the paraoxonase gene locus (PON1) are the leucine (L allele)→methionine (M allele) and the glutamine (Q allele)→arginine (R allele) substitutions at residues 55 and 192, respectively. We have examined the influence of these two polymorphisms on carotid atherosclerosis in familial hypercholesterolemia (FH) patients. The allele frequencies of these two polymorphisms were determined by PCR and restriction fragment analysis, for both the FH population and healthy controls. High resolution B-mode ultrasound was used to assess intima-media wall thickness (IMT) of the carotid artery. No differences were found in allele frequencies between the FH and the control population. In FH patients, the LL, LM and MM genotypes at position 55 occurred in 86 (46.0%), 78 (41.7%) and 23 (12.3%) subjects, respectively, whereas the QQ, QR and RR genotypes at position 192 were found in 90 (48.1%), 79 (42.2%) and 18 (9.6%) individuals. When both polymorphisms were considered separately, no different carotid IMTs were found between the genotype groups. However, our data did show a significant association between the various genotypes of the combined polymorphisms at position 55 and 192 of PON1 and the carotid artery IMT in FH subjects. Subjects with the homozygous wildtype LL/QQ for paraoxonase had the highest mean carotid IMTs when compared to other genotypes, combined. Multiple regression analysis demonstrated age (β=0.34, P<0.0001), total plasma cholesterol (β=0.17, P=0.0109) and the LL/QQ genotype of the PON1 gene (β=0.22, P=0.0018) to be significant risk factors for carotid atherosclerosis in subjects with FH. The LL/QQ genotype could explain 5.3% of total variance of carotid IMT. In conclusion, this is the first study to report an independent association between the combined PON1 polymorphism genotypes and carotid wall thickness. The homozygous wildtype LL/QQ for PON1 may represent an additional risk factor for carotid atherosclerosis in subjects with FH.

Lipoprotein (a) [Lp(a)] is a quantitative genetic trait in human plasma and elevated levels represent a major inherited risk factor for the development of atherosclerotic disease. In our search for sequence polymorphisms in the coding region of the apolipoprotein(a) [apo(a)] gene that may affect the Lp(a) concentration, four new polymorphic sites were identified. These include two coinciding polymorphisms with an allele frequency of 38% located at amino acid positions 87 and 101 (Leu87,101-->Val) in the interkringle region of kringle IV (K.IV) type 7 and two polymorphisms located in K.IV type 7 (Arg60-->Ser) and in K.IV type 10 (Tyr2-->Phe) both with estimated allele frequencies of about 1%. The linkage between the newly identified K.IV type 7 Leu87,101 -->Val polymorphism and earlier described polymorphic sites in the non-coding and coding regions of the apo(a) gene, its distribution over the apo(a) isoform sizes and its possible influence on the Lp(a) concentration was analysed in 201 healthy unrelated Caucasians. The earlier described polymorphic sites included in this study were the variable number of a TTTTA pentanucleotide repeat (7-11 PNR) starting at -1231 bp, the -772 bp G/A polymorphism, the +93 bp C/T polymorphism and the +121 bp G/A polymorphism in the non-coding region, and the K.IV type 8 Thr12/Pro polymorphism and the K.IV type 10 Thr66/Met polymorphism in the coding region of the apo(a) gene. Linkage disequilibria were observed between the polymorphic sites in the 5' non-coding region and the sites in K.IV type 7 and 8 in the coding region of the apo(a) gene, confirming that the expansion of the variable number of K.IV type 2 repeats results from intrachromosomal recombinational events. The distribution over the apo(a) isoform sizes of the K.IV type 7 Val87,101 subtype was not significantly different from that of the K.IV type 7 Leu87,101 wild-type, suggesting a relative ancient mutational event. No influence of the K.IV type 7 Leu87,101-->Val polymorphism on the Lp(a) level was observed. In fact, of all the polymorphic sites studied, only the +121 A subtype could be associated with an increased, and the K.IV type 8 Pro12 and the 10 PNR subtypes with a reduced, Lp(a) concentration corrected for apo(a) isoform size (p <0.05).

Low vitamin D status may be involved in development of osteoporosis. Augmentation of vitamin D status by exposure to UV-B can not cause vitamin D toxicity, but carries the risk of skin cancer development. Oral vitamin D supplementation of postmenopausal women living at high latitudes in winter is therefore recommended. There is concern that this strategy may cause vitamin D toxicity. Little is known about the buffering role of adipose tissue in vitamin D storage and release. The aim of this study was to investigate vitamin D storage in rat adipose tissue, and its subsequent unstimulated and stimulated release. Female Wistar rats were supplemented with 1500 IU vitamin D3/day during 14 days. Plasma vitamin D3 and 25-hydroxyvitamin D [25(OH)D], and adipose tissue vitamin D3 were monitored during 99 days. From day 14 a subgroup of rats was fasted during 72 hours to investigate the effect of stimulated vitamin D release from adipose tissue. Following vitamin D supplementation, plasma vitamin D3 reached steady state levels within 3 days. 25(OH)D increased more slowly to reach plateau levels from about day 10. Adipose tissue vitamin D3 rose linearly until day 14. Following discontinuation of vitamin D3 supplementation, plasma vitamin D3 decreased more quickly (t1/2=29 hours) than 25(OH)D (t1/2=18 days). There were no changes in adipose tissue vitamin D3 contents. Fasted rats lost about 10% weight, but did not show different courses of plasma vitamin D3 and 25(OH)D, compared with ad libitum fed counterparts. Their adipose vitamin D3 content (in nmol/g wet weight) rose, indicating that fatty acid mobilization from adipose tissue occurs more easily than that of vitamin D. We conclude that orally supplemented vitamin D rapidly accumulates in adipose tissue, but slowly releases following discontinuation. Storage capacity seems unsaturable and fasting does not cause its massive release. Uptake of vitamin D in adipose tissue may be an important factor in the prevention of vitamin D toxicity and maintenance of long term adequate vitamin D status. Lipiden

De bepaling van vet in faeces wordt tot nu toe binnen onze laboratoria uitgevoerd volgens de 'van de Kamermethode'. Deze methode is zeer bewerkelijk, tijdrovend en een kwaliteitscontrole ontbreekt. In samenwerking met twee andere academische centra (Groningen en Utrecht) hebben we een nieuwe vet in faecesbepaling ontwikkeld die gebruik maakt van mid-infraroodspectroscopie. Deze techniek wordt binnen de klinische chemie al gebruikt voor de niersteenanalyse. Na een korte en eenvoudige voorbewerking van de faecesmonsters, waarbij de vetzuren geïsoleerd worden uit de faeces m.b.v. een aangezuurd mengsel van petroleumether en ethanol, werd een transmissiespectrum opgenomen in het mid-infraroodgebied (400 4000 cm-1). Met behulp van 'Partial Least Square' en multicomponentanalyses van de golflengten, gemeten bij diverse faecesmonsters met een bekende vetconcentratie, werd een model gegenereerd. Tevens werd stearinezuur gebruikt als standaard voor de ijklijn. Er bleek een goede correlatie te zijn tussen de vetconcentraties bepaald met infrarood en gemeten met de 'van de Kamermethode' (n=35, r2> 0,95). Conclusie: de bepaling van vet in faeces met behulp van mid-infraroodspectroscopie biedt, in zijn eenvoud en standaardisatiemogelijkheden, een goed alternatief voor de conventionele 'van de Kamermethode'. Lipiden

Lipoprotein(a) [Lp(a)], an independent risk factor for the development of atherosclerosis, contains an apolipoprotein(a) [apo(a)] moiety covalently linked to a LDL moiety. Apo(a) is a glycoprotein homologous to plasminogen as it contains multiple repeats of a lysine binding domain resembling plasminogen kringle IV (K.IV). The multiple K.IV repeats can be differentiated in ten types that show a variation in their lysine binding capacity. Since K.IV type 10 shows the highest conservation of the amino acids postulated to form the lysine binding pocket, this kringle is suggested to be the main lysine binding site of apo(a). Recently, a T-->C polymorphism in the apo(a)-gene was reported, leading to a Met-->Thr substitution at amino acid position 66 of K.IV type 10, in the vicinity of the postulated lysine binding pocket. To investigate the significance of this substitution on some in vitro characteristics of Lp(a), the affinity for lysine-Sepharose and the binding affinity for limited plasmin digested des AA fibrin (Desafib-X) of the two subtypes was determined using plasma of donors homozygous for the polymorphism. These studies revealed a large heterogeneity in the binding characteristics, irrespective of the subtype. The comparison of the allele frequencies of this polymorphism in 155 patients having symptomatic atherosclerosis versus 153 normolipidemic controls revealed no significant differences. In conclusion, this study suggests that the presence of either a Met66 or a Thr66 residue in K.IV type 10 of apo(a) has no consequences for the binding characteristics of Lp(a) toward lysine-Sepharose or Desafib-X, nor is it associated with the presence of symptomatic atherosclerosis.

Elevated plasma levels of lipoprotein(a) [Lp(a)] represent a significant independent risk factor for the development of atherosclerosis. Interindividual levels of apo(a) vary over 1000-fold and are mainly due to inheritance that is linked to the locus of the apolipoprotein(a) [apo(a)] gene. The apo(a) gene encodes multiple repeats of a sequence exhibiting up to 85% DNA sequence homology with plasminogen kringle IV (K.IV), a lysine binding domain. In our search for sequence polymorphisms in the K.IV coding domain, we identified a polymorphism predicting a Thr-->Pro substitution located at amino acid position 12 of kringle IV type 8 of apo(a). The functional and clinical significance of this polymorphism was analysed in a case-control study and by comparing the in vitro lysine binding characteristics of the two Lp(a) subtypes. The case-control study (involving 153 subjects having symptomatic atherosclerosis and 153 age and gender matched normolipidemic controls) revealed a overall allele frequency for the Thr12-->Pro substitution in kringle IV type 8 of 14% and a negative association between presence of the Pro12-subtype and symptomatic atherosclerosis (p < 0.03). The in vitro lysine binding studies, using Lp(a) isolated from subjects homozygous for either Thr12 or Pro12 in K.IV type 8, revealed comparable lysine-Sepharose binding fractions for the two subtypes. The binding affinity (Kd) for immobilised plasmin degraded des-AA-fibrin (Desafib-X) was also comparable for the two subtypes, however a decreased maximal attainable binding (Bmax) for immobilised desafib-X was observed for the Pro12-subtype Lp(a).

Elevated plasma levels of lipoprotein (a) (Lp(a)) in humans represent a major inherited risk factor for atherosclerosis. Lp(a) consists of a LDL-like particle with an additional glycoprotein, apolipoprotein(a) (apo(a)), linked to apolipoprotein B100. The apo(a) moiety is highly homologous to plasminogen as it contains multiple repeats resembling plasminogen kringle IV, a lysine and fibrin binding domain. In the present study, the individual contribution of the two constituents of Lp(a), namely LDL and apo(a), in the binding of Lp(a) to limited plasmin digested des AA fibrin (Desafib-X) is examined.Lp(a) was isolated by sequential ultracentrifugation followed by gel filtration chromatography. Lysine binding (Lp(a)lys') and non-lysine binding (Lp(a)lys(-)) Lp(a) were obtained by affinity chromatography using lysine-Sepharose. Lp(a)-free LDL was isolated by ultracentrifugation followed by chromatofocusing. I-125-labelled Lp(a), LDL, Lp(a)lys(-), and Lp(a)lys(+) preparations were incubated with Desafib-X coated wells in the presence or absence of epsilon-aminocaproic acid (epsilon ACA, a lysine-analogue) and/or autologous LDL.Total Lp(a) contained 86 +/- 8% Lp(a)lys(+). The mean apparent dissociation constant (Kd in nM) for Lp(a), LDL, Lp(a)lys(-), and Lp(a)lys(+) binding to Desafib-X was 48 +/- 11, 28 +/- 4, 7 +/- 4, and 43 +/- 23, respectively. The binding of Lp(a) and Lp(a)lys(+) to Desafib-X could be inhibited to a similar degree by 0.2 m epsilon ACA (for 31 +/- 14% and 37 +/- 15% respectively), indicating lysine specific binding of these preparations. The binding of Lp(a)lys(-) and LDL could not be inhibited by epsilon ACA. A ten times molar excess of LDL could inhibit the binding of Lp(a), Lp(a)lys(-), and Lp(a)lys(+) to Desafib-X by 60 to 80%. For Lp(a) and Lp(a)lys(+), an additional binding-inhibition can be observed when adding 0.2 M epsilon ACA.In conclusion, the overall findings indicate that the binding of Lp(a) to fibrin(-ogen) is more complex than previously thought and imposes an influence of the LDL moiety and LDL, additional to the apo(a) moiety, in the binding of Lp(a) to lysine-containing proteins like Desafib-X.

Lipoprotein(a) (Lp(a)) is a LDL-like particle with an additional glycoprotein, apo(a), linked to apolipoprotein B-100. Apo(a) is highly homologous to parts of the plasminogen molecule, and numerous investigations have shown interference of Lp(a) with functions of plasminogen. In this report we studied the influence of apo(a) phenotype on the binding of Lp(a) to plasmin-modified immobilized des-AA-fibrinogen (desafib-X).Results indicate that Lp(a) binds to desafib-X in a specific and saturable way. There was a strongly significant negative correlation between apo(a) isoform length and maximal number of Lp(a) particles bound to the desafib-X matrix (N=18, r=0.84, p=0.002). There was no relation between apo(a) isoform length and K-d for the binding of Lp(a) to desafib-X. In two donors (10%) no specific binding to desafib-X was observed, as well as to lysine-sepharose.In conclusion, apo(a) isoform length influences the amount of Lp(a) binding to desafib-X. This implies that small isoforms apart from their usual higher plasma Lp(a) concentrations also are potentially more thrombogenic. The fact that Lp(a) from two donors did not bind to desafib-X, suggests that mutations may exist in the K4-10 domain of apo(a) abolishing its lysine-binding ability.

Three commercially available assays (an enzyme-linked immunosorbent assay ELISA, an immunoradiometric assay, IRMA, and a nephelometric assay) for the determination of lipoprotein(a) [Lp(a)] were compared with respect to the dependency of these assays on the various apolipoprotein(a) [apo(a)] isoforms. Although there was a strong correlation between the three methods, a significant difference between the absolute values (mg/L) was observed (p < 0.001). Using purified Lp(a) preparations, we showed that the ELISA assay quantifies the Lp(a) concentration on a molar basis, independently of the apo(a) isoform size. The IRMA and the nephelometric assay however are apo(a) isoform size dependent and overestimate the Lp(a) concentration of large apo(a) isoforms whereas the amount of small apo(a) isoforms is underestimated. In general, the isoform dependency of the Lp(a) quantification is of limited clinical relevance. In this study, inconsistent risk assignments are made in approximately 3% of the cases, when the Lp(a) concentrations obtained with the apo(a) isoform dependent assays are compared with the isoform independent ELISA.