Cholesterol metabolism in cells with different peroxisomal defects.

We showed previously that cholesterol biosynthesis in dermal fibroblasts from patients with metabolic disorders of peroxisomal origin is increased in steps prior to mevalonate, whereas low-density-lipoprotein(LDL)-receptor activities were not different from control fibroblasts. Here, the suppression of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase activity by lovastatin was studied both in dermal fibroblasts from patients with different peroxisomal defects and in a Chinese hamster ovary (CHO) cell line lacking morphologically intact peroxisomes. In addition, the formation of intracellular cholesteryl esters (a measure of acyl-CoA:cholesterol acyltransferase(ACAT)-activity) stimulated by exogenous LDL was investigated. A dose-dependent suppression of cholesterol biosynthesis by lovastatin at concentrations of 1-50 mumol/l was observed which was comparable in normal and peroxisomal-disease fibroblasts. ACAT activity was measured in the absence and presence of exogenous LDL using [3H]oleate as a substrate for cholesterol ester synthesis. The basal esterification rate was equal or lower in peroxisomal-defective fibroblasts compared with controls. In the presence of exogenous LDL, cholesterol esterification was significantly impaired in all defective cells in comparison with normal fibroblasts. We conclude that changes in cholesterol homeostasis in peroxisomal diseased fibroblasts be related to cholesterol ester formation.

Immunoturbidimetric determination of lipoprotein(a): improvement in the measurement of turbid and triglyceride-rich samples.

Immunoturbidimetry (IT), a widely used method in clinical chemical laboratories, was checked for its suitability for lipoprotein(a) (Lp(a)) quantification. When the conventional sample diluents were used, turbidimetry gave false results particularly with frozen or lipemic sera which correlated poorly with electroimmunodiffusion (EID). L-Proline which is known to dissociate Lp(a) from other apo B-containing lipoproteins improved the results considerably. One hundred frozen sera were investigated in IT with and without the addition of L-proline to the sample diluent. EID served as a comparison method. In a method comparison (IT vs. EID) linear regression analysis improved from r = 0.793: y = 0.89x - 9.4 (without L-proline) to r = 0.949: y = 0.98x + 4.8 (with L-proline). The improvement of the correlation of the two methods was most pronounced in sera with triglyceride values exceeding 5.5 mmol/l. The IT assay described here was linear between 50 and 1100 mg/l. Total imprecision (coefficient of variation) was below 10%. The assay was not affected by the addition of LDL or plasminogen to the samples. The Lp(a) concentration of the calibrator, i.e. a secondary standard serum, was compared with that of a purified primary Lp(a) standard which consisted of a mixture of four apo(a) isoforms. Total Lp(a) mass (lipids, protein, carbohydrates) was determined chemically and was compared with the Lp(a) mass determined immunochemically by IT and EID. Recovery of the purified Lp(a) was 106% (range 90-116%) in IT and 102% (range 91-115%) in EID. Dose response curves from pure single isoforms (S1 and S4), calibrator and serum samples were parallel. We consider IT to be a simple and rapid method for Lp(a) quantification which is not biased by different apo(a) isoforms.

The interaction of Lp(a) with normal and LDL-receptor-deficient human skin fibroblasts.

The role of LDL receptors in the in vivo catabolism of Lp(a) is still a matter of controversy. Since Lp(a) binds LDL with high affinity, it was essential for this study to separate Lp(a) quantitatively from all other apo-B and apo-E-containing lipoproteins. This was achieved by the addition of proline as a dissociating agent to all buffers during Lp(a) preparation. Normal human skin fibroblasts pre-incubated with 40 mg/ml of Lp(a) downregulated cholesterol biosynthesis by approx. 35%; the same amount of LDL caused a 90% reduction. Cholesterol biosynthesis of LDL-receptor-deficient fibroblasts was not affected at all by LDL, yet Lp(a) exhibited a similar effect as in normal fibroblasts (32% reduction). An LDL-receptor-independent uptake of Lp(a) into fibroblasts must therefore be postulated. We also studied the degradation of Lp(a) in normal fibroblasts in comparison with LDL. Pure Lp(a) was only slightly degraded in relation to LDL. If fibroblasts were pre-incubated with small amounts of LDL, Lp(a) degradation was enhanced by a factor of 3-5. This effect was even more pronounced in fibroblasts pre-incubated with mevinolin. Thus the LDL receptor may play an indirect role in Lp(a) catabolism. The significance of these findings for the in vivo metabolism of Lp(a) remains to be established.

Lipoprotein(a) mediates high affinity low density lipoprotein association to receptor negative fibroblasts.

Lipoprotein(a) (Lp(a)) is an acute phase protein with unknown function. Lp(a) binds to low density lipoprotein (LDL) receptors, as well as to plasminogen (Plg) receptors. Preincubation of normal human skin fibroblasts with Lp(a) or with apo(a) cause a severalfold increase of LDL binding. Plg and kringle-4 of Plg have no effect. LDL receptor-negative fibroblasts respond upon preincubation with apo(a) with high affinity binding of LDL with Kd values that are almost identical with those of LDL binding to the LDL receptor. Incubation of apo(a)-pretreated fibroblasts with anti-apo(a) completely abolishes the increment of LDL binding. The high affinity LDL binding to LDL receptor-negative fibroblasts could be dissociated by approximately 80 and 54% with 5 mg/ml proline and 30 mg/ml NaCl, respectively, but not with dextran sulfate. The Lp(a)- and apo(a)-triggered LDL binding to fibroblasts have no effect on LDL internalization. These findings may reflect a key function in the role as an acute phase protein and may be relevant to the high atherogeneicity of Lp(a).

Preparation of a stable fresh frozen primary lipoprotein[a] (Lp[a]) standard.

LP[a] is one of the most atherogenic lipoproteins consisting of an LDL-like core particle and a covalently linked glycoprotein of variable size. Due to its structural features, its heterogeneity and instability, there are great difficulties in standardizing quantitative immunochemical Lp[a] assays. One particular problem is the preparation of a pure primary standard, which is sufficiently stable to be used for value assignment of secondary reference material. Here we describe a method to purify Lp[a] to virtual homogeneity. When mixed with glycerol at a ratio of 1:1, the preparation is stable in the deep frozen state for more than 12 months. This latter material gave dose;-response curves in several immunochemical assays that were parallel to fresh or frozen sera, freshly prepared Lp[a], and other proposed reference materials. After determination of the protein content by amino acid analysis, it was possible to assign concentrations in molar and mass units to these preparations considering the theoretical molecular weights of the particular apo[a] isoform. Thus we propose to use this procedure for preparation of a "gold standard" for Lp[a] assays.

Organization of phosphatidylcholine and sphingomyelin in the surface monolayer of low density lipoprotein and lipoprotein(a) as determined by time-resolved fluorometry.

Fluorescent analogs of phosphatidylcholine (PC) and sphingomyelin (SM) labeled with diphenylhexatrienylpropionic acid (DPH) were prepared and incorporated into the surface layer of human low density lipoprotein (LDL) and lipoprotein(a) (Lp(a)). Fluorescence anisotropy measurements of DPH-PC and DPH-SM in both lipoprotein classes were carried out at different temperatures ranging from 20 to 37 degrees C. DPH-PC as well as DPH-SM were shown to reside in more rigid domains in Lp(a) than in LDL according to higher anisotropy values in Lp(a). In both LDL and Lp(a), DPH-PC experienced a more rigid environment than DPH-SM, suggesting different environments of PC and SM in the surface shell of the lipoproteins. Fluorescence lifetimes of the labeled lipoproteins were determined by phase and modulation fluorometry. We found bimodal Lorentzian distributions for the decay times of DPH-PC and DPH-SM in LDL and Lp(a). Lifetime distribution centers for labeled lipids were very similar except for DPH-PC in Lp(a) which was shifted to longer lifetimes, suggesting a less polar environment of PC in Lp(a) than in LDL. The distributional width of DPH-PC in Lp(a) was broader than in LDL. Accordingly, phosphatidylcholine must be localized in a more homogeneous environment in LDL as compared with Lp(a). On the other hand, no difference in distributional widths was observed for DPH-SM in both lipoproteins, showing that SM organization in Lp(a) is unaffected by apo(a). From the obtained fluorescence data we propose that apoproteins discriminate between the choline phospholipids and preferentially associate with phosphatidylcholine. This effect is enhanced in Lp(a) due to the presence of apolipoprotein(a).

Transfer of phospholipase A-resistant pyrene-dialkyl-glycerophosphocholine to plasma lipoproteins: differences between Lp[a] and LDL.

1-O-Hexadecyl-2-O-pyrenedecanyl-sn-glycero-3-phosphocholine, a non-hydrolyzable fluorescent diether analog of phosphatidylcholine (PC), was synthesized as a probe for studying phospholipid transfer to different lipoprotein classes with potential phospholipase activities. After incubation of total human plasma with the new probe at 37 degrees C for 4.5 h, a characteristic partition between the main lipoprotein fractions was observed. The fluorescent lipid was not degraded under these conditions and, therefore, served as a measure for choline glycerophospholipid distribution between plasma lipoproteins. In low density lipoprotein (LDL) and high density lipoprotein-3 (HDL3) the fluorescent PC analog showed only monomer fluorescence, whereas in Lp[a] and HDL2 monomer and excimer fluorescence were observed, indicating that the fluorescent phosphatidylcholine analog was incorporated into the respective lipoproteins to a different extent. According to the increased pyrene excimer fluorescence in Lp[a] compared with LDL the labeled phosphatidylcholine must be enriched and/or clustered in Lp[a]. Data from phospholipid and total fluorescence analyses are compatible with the assumption of higher label concentration in Lp[a]. On the other hand, transfer rates for serum protein-catalyzed lipid transport into isolated Lp[a] were slower as compared to LDL. It is suggested that slower lipid transfer to Lp[a] under these conditions is due to the decreased lipid mobility in the Lp[a] surface, whereas the higher extent of label partition into Lp[a] as observed in total plasma might be due to the higher affinity of apolipoproteins for phosphatidylcholine in Lp[a] (Sommer, A., et al. 1992. J. Biol. Chem. 267: 24217-24222). The use of a fluorescent dialkyl- instead of diacyl-glycerophosphocholine for transfer studies was mandatory, as we found that lipoproteins contained phospholipase A2 activity toward long-chain phosphatidylcholine. The lipoprotein-associated phospholipase A2 was three times more active in Lp[a] than in LDL. The degradation products formed by the phospholipase, fatty acids, and lyso-PC may add to the high atherogenic potential of Lp[a].