Mouse serum paraoxonase-1 lactonase activity is specific for medium-chain length fatty acid lactones

Recent studies suggest that paraoxonase-1 (PON1), complexed with high-density lipoproteins, is the major lactonase in the circulation. Using 5-hydroxy eicosatetraenoate δ-lactone (5-HETEL) as the substrate, we observed lactonase activity in serum from Pon1-/- mice. However, 6-12 carbon fatty acid γ- and δ-lactones were not hydrolyzed in serum from Pon1-/- mice. Serum from both wild-type and Pon1-/- mice contained a lactonase activity towards 5-HETEL and 3-oxo-dodecanoyl-homoserine lactone that was resistant to inactivation by EDTA. This lactonase activity was sensitive to the serine esterase inhibitor phenyl methyl sulfonyl fluoride and co-eluted with carboxylesterase activity by size-exclusion chromatography. Analysis of serum from the Es1e mouse strain, which has a deficiency in the carboxylesterase, ES-1, proved that this activity was due to ES-1. PON1 activity predominated at early time points (30 s), whereas both PON1 and ES-1 contributed equally at later time points (15 min). When both PON1 and ES-1 were inhibited, 5-HETEL was stable in mouse serum. Thus, while long-chain fatty acid lactones are substrates for PON1, they can be hydrolyzed by ES-1 at neutral pH. In contrast, medium-chain length fatty acid lactones are stable in mouse serum in the absence of PON1, suggesting that PON1 plays a specific role in the metabolism of these compounds.     

Paraoxonase 1 (PON1) and its relationship to lipid variables, age and gender in healthy volunteers

Recent studies implied that low-density lipoprotein (LDL) modified predominantly by oxidation or glycation, significantly contributes to the formation of atherosclerotic lesions. In contrast to oxidized LDL (ox-LDL), high-density lipoprotein (HDL) is able to prevent accumulation of ox-LDL in arterial walls. This antiatherogenic property of HDL is attributed in part to several enzymes associated with the lipoprotein, including HDL-associated paraoxonase 1 (PON1). In this study we analyzed PON1 arylesterase/paraoxonase activities in relation to serum lipid profile, gender and age in thirty clinically healthy Slovak volunteers. Our results showed that PON1 arylesterase and paraoxonase activities were lower in citrated plasma than in serum by 16.6% and 27.3%, respectively. Among serum lipoproteins, only HDL-cholesterol level showed significant positive correlation with PON1 arylesterase activity (p = 0.042). Likewise, we found a significant relationship between atherogenic index (AI = total cholesterol/HDL-cholesterol) and PON1 arylesterase activity (p = 0.023). No significant correlation could be demonstrated between PON1 paraoxonase activity and serum lipid profile, age or gender. Furthermore, it was found that PON1 paraoxonase/arylesterase activities were higher in women compared with both investigated activities in men, but these differences were not statistically significant. These results confirmed a positive correlation between HDL-cholesterol and PON1 arylesterase activity. Moreover, it was found out that PON1 paraoxonase activity is not influenced either by gender or by age. PON1 arylesterase activity was however affected by gender to a limited extent.     

The determination of Q192R polymorphism of paraoxonase 1 by using non-toxic substrate p-nitrophenylacetate

CONTEXT:

The human serum paraoxonase 1 (PON1) is calcium-dependent esterase and associates with the high density serum lipoproteins. PON1 plays a major role in oxidation of high density lipoprotein and low density lipoprotein and prevention of atherogenesis in coronary heart disease. PON1Q and R allele hydrolyses number of substrates like paraoxon (PO) (diethyl p-nitrophenyl phosphate) and phenylacetate.

AIMS:

The aim of the study is to the determination of Q192R polymorphism of PON1 by using non-toxic substrate p-nitrophenylacetate and compares it with the phenotype determined by using PO as substrate.

MATERIALS AND METHODS:

The study group consists of 60 healthy normal patients. Paraoxonase activity was measured using the procedure described by Eckerson (Reference method) and for phenotyping; the ratio of hydrolysis of PO in the presence of 1 M NaCl (salt-stimulated PON1, SALT) to the hydrolysis of phenylacetate (PA) is calculated. In new method (Haagen et al.) arylesterase activity measured using p-nitrophenylacetate and for phenotyping arylesterase, the ratio of inhibition of enzymatic hydrolysis of p-nitrophenylacetate (substrate) by phenyl acetate to non-inhibited hydrolysis of p-nitrophenylacetate (inhibited arylesterase activity (IA-IA₀)/non-inhibited arylesterase activity (NIA).

RESULTS:

It was found that paraoxonase activity is trimodally distributed in both the methods. There is no significant difference in the distribution of PON1 phenotypes of both reference method and new method being frequencies 0.946 and 0.376 respectively and there was no significant difference for phenotypic polymorphism for an individual by both methods (χ²= 0.15 and P = 0.9262).

CONCLUSION:

The Q192R polymorphism of PON1 by using non-toxic substrate p-nitrophenylacetate showed trimodal distribution of QQ (homozygous), QR (heterozygous), and RR (homozygous) phenotype and it is comparable with reference method. This method can be used for PON1 phenotype in different pathological and complex disease conditions.     

Levels of paraoxonase and arylesterase activities and malondialdehyde in workers exposed to ionizing radiation

We examined levels of malondialdehyde (MDA) (an end-product of lipid peroxidation) and paraoxonase (PON1) (an antioxidant enzyme) activity and PON1 phenotypes in people who were exposed to ionizing radiation for different time periods and doses. A total of 78 individuals (mean age 34 +/- 7 years) were included in the study. Fifty-one of them were radiology workers whereas the control group was composed of 27 healthy volunteers who had never worked in a radiology-related job. Paraoxon was used as substrate for measurement of PON1 activity levels (basal and NaCl-stimulated). Phenylacetate was used as substrate for measurement of arylesterase activity levels. Cumulative levels of serum NaCl-stimulated PON1/arylesterase activities were utilized for phenotypic differentiation. In radiology workers, three different phenotypes were determined based on paraoxonase/arylesterase ratio. The ratios were 1.09 +/- 0.30 for AA (homozygote low activity); 2.91 +/- 1.07 for AB (heterozygote activity) and 4.97 +/- 1.21 for BB (homozygote high activity). There was a statistically meaningful negative correlation between serum MDA levels and PON1 activity levels in all phenotypes (p < 0.05). PON1 activity levels were found to be 25-35% lower in people who were exposed to long-term ( > 5 years) radiation compared to controls. There was no statistically significant correlation between serum arylesterase activity and MDA levels in these subjects (r = -0.185, p > 0.05). PON1 activity levels were decreased whereas serum MDA levels were increased in individuals exposed to radiation for a long period. PON phenotypes of people employed in jobs which expose them to radiation should be determined and based on these findings they should be advised to avoid risk factors inducing oxidative stress, such as smoking, and to consume foods rich in vitamins and trace elements to increase their antioxidant capacity.     

Presence and isolation of 2 lipoproteins immunologically related to apolipoprotein A I in human serum

Very high density lipoproteins d : 1.23--1.25 g/ml (VHDL2) have been isolated from human serum by preparative ultracentrifugation. They contain 80 per cent proteins and 20 per cent lipids. Lipids are mainly phospholipids (80 per cent). The proportion of lysolecithin (50 per cent) is higher than that of lecithin (40 per cent). The quantity of cholesterol is low, the free cholesterol: total cholesterol ratio is 0.35. VHDL2 consisted principally in lipoprotein D and two lipoproteins immunologically apparented to apolipoprotein A I, called LP A I1 and LP A I2. The LP A I1 has a molecular weight slightly higher and a hydrated density lower than that of LP AI2. Our experiments suggest that LP A I1 exists in the serum before ultracentrifugation while LP A I 2 comes from HDL degradation during ultracentrifugation. The immunological heterogeneity of apo A I forming different protein-lipid complexes is discussed.     

Distribution and characterization of the serum lipoproteins and apoproteins in the mouse, Mus musculus

Murine lipoproteins were separated into nine subfractions by a density gradient ultracentrifugal procedure. They were characterized by electrophoretic, immunological, chemical, and morphological analyses, and their protein moieties were defined according to charge, molecular weight, and isoelectric point. HDL predominated (approximately 500 mg/dl serum), the mode of its distribution being situated in the d 1.09-1.10 g/ml (F 1.21 approximately 4) region. Chemical analysis showed subfractions of d 1.085-1.136 g/ml to resemble human HDL3 closely, including the presence of apoA-I (Mr 25,000-27,000) as their major apolipoprotein. An apoA-II-like protein, of Mr 8400 (in monomeric form), was also tentatively identified. In electrophoretic mobility and chemical composition, the d 1.060-1.085 g/ml subfraction (approximately 10% of total HDL) was distinct and akin to human HDL2. ApoA-I represented approximately 60% of its complement of low molecular weight apoproteins. The density range used for separation of human HDL2 (d 1.066-1.100 g/ml) by gradient ultracentrifugation is inadequate in the mouse, and the d 1.060-1.085 g/ml interval is more appropriate. The 1.063 g/ml boundary for separation of mouse LDL from HDL was unsuitable. Immunological and electrophoretic studies revealed that alpha-migrating lipoproteins were present in the d 1.046-1.060 g/ml range, a finding consistent with their enrichment in apoA-I; apoE-, apoA-II-, and apoC-like proteins were also detected. These findings indicate the presence of HDL1 particles. Murine apoA-I and apoB-like proteins of higher (apoBH) and lower (apoBL) molecular weight were constituents of the d 1.033-1.046 g/ml fraction. Alternative techniques, such as electrophoresis in starch block, are therefore a prequisite for separation of apoB from alpha-migrating, apoA-I-containing lipoproteins in the low density range in mouse serum. The LDL class (d 1.023-1.060 g/ml) amounted to only approximately 20% of the total murine lipoproteins of d less than 1.188 g/ml (65-70 mg/dl serum). Particles were richer In triglyceride, larger in diameter (mean 244 A), and more heterogeneous than typical of man. VLDL (40-80 mg/dl serum) was triglyceride-rich (66% by weight) and similarly heterogeneous in size (mean diameter 494 A; range 270-750 A). ApoBH and apoBL were prominent in murine VLDL, and cross-reacted with an antiserum to human apoB. ApoE- and apoA-I-like proteins were also detectable in apoVLDL, as was a protein of 70,000-75,000 mol wt. The presence of murine apolipoproteins analogous to human apoB and apoE was confirmed by the immunological cross-reactivities of VLDL and LDL with monospecific antisera to the human proteins. The marked similarity of lipoprotein and apolipoprotein profile in the mouse and rat is notable. Since murine VLDL contains apoE and apoBL, this resemblance may extend to the metabolism of chylomicron remnants and hepatic VLDL in the two species.     

Early changes in plasma lipoprotein structure and biosynthesis in cholesterol-fed rabbits

Plasma lipoproteins of d < 1.063 g/ml from rabbits fed a diet containing 1% cholesterol for 4 days showed changes in concentration and rates of flotation as determined by analytical ultracentrifugation. A marked increase in cholesteryl ester content of lipoprotein with d < 1.019 g/ml was the most prominent change in rabbits fed the diet for 21 days. Gel electrophoresis and immunochemical procedures demonstrated that in control and hypercholesterolemic rabbits there were some common apolipoproteins found in all lipoproteins with density < 1.063 g/ml. In control rabbits, there were also apolipoproteins specific to the lipoprotein fraction with d < 1.019 and to the fraction with d 1.019-1.063 g/ml. However, in rabbits fed the hypercholesterolemic diet for 21 days, the apolipoproteins characteristic of fraction 1.019-1.063 were the most abundant in the fraction with d < 1.019 g/ml. Liver slices from rabbits fed the high cholesterol diet for 7 and 21 days incorporated more l-[(14)C]leucine into very low density and low density lipoproteins than controls. The results suggest that cholesterol feeding leads to an increase in biosynthesis of lipoproteins with d < 1.063 g/ml. The newly synthesized lipoprotein contains apolipoproteins similar to those found in controls but with a higher lipid-to-protein ratio. From the apoprotein composition, it is concluded that the very low density fraction present in cholesterol-fed animals is more structurally related to low density lipoproteins than to the very low density lipoproteins isolated from control animals.     

A sandwich enzyme-linked immunosorbent assay for human serum paraoxonase concentration

Serum paraoxonase (PON) is associated with plasma high density lipoproteins, and prevents the oxidative modification of low density lipoproteins. We have developed a sensitive sandwich enzyme-linked immunosorbent assay (ELISA), using two monoclonal antibodies against PON, to measure serum PON concentration. The concentration of PON in healthy Japanese subjects was 59.3 +/- 1.3 microgram/mL (mean +/- SEM; n = 87). Serum PON concentrations in relation to the PON 192 genetic polymorphism were: 69.5 +/- 2.9 microgram/mL in the QQ genotype; 63.0 +/- 1.9 microgram/mL in the QR genotype; and 52.8 +/- 1.7 microgram/mL in the RR genotype. Concentrations were significantly lower in the RR than in the QQ genotype (P < 0.01). Serum paraoxonase specific activity was higher in RR than in QQ subjects (18.6 +/- 0.40 vs. 2. 56 +/- 0.05 nmol/min/microgram, P < 0.01), but arylesterase specific activity was unrelated to genotype. PON concentration was positively associated (P < 0.001) with both serum arylesterase activity and, after adjusting for the effect of the position 192 polymorphism, with serum paraoxonase activity. Subjects with angiographically verified coronary heart disease had significantly lower PON concentrations than the healthy controls (52.0 +/- 2.3 microgram/mL; n = 35, P < 0.01). This association was independent of the position 192 genotype. Our new ELISA should be of value for epidemiologic and clinical studies of serum PON concentration. immunosorbent assay for human serum paraoxonase concentration.     

Rabbit serum paraoxonase 3 (PON3) is a high density lipoprotein-associated lactonase and protects low density lipoprotein against oxidation

The paraoxonase gene family contains at least three members: PON1, PON2, and PON3. The physiological roles of the corresponding gene products are still uncertain. Until recently, only the serum paraoxonase/arylesterase (PON1) had been purified and characterized. Here we report the purification, cloning, and characterization of rabbit serum PON3. PON3 is a 40-kDa protein associated with the high density lipoprotein fraction of serum. In contrast to PON1, PON3 has very limited arylesterase and no paraoxonase activities but rapidly hydrolyzes lactones such as statin prodrugs (e.g. lovastatin). These differences facilitated the complete separation of PON3 from PON1 during purification. PON3 hydrolyzes aromatic lactones and 5- or 6-member ring lactones with aliphatic substituents but not simple lactones or those with polar substituents. We cloned PON3 from total rabbit liver RNA and expressed it in mammalian 293T/17 cells. The recombinant PON3 has the same apparent molecular mass and substrate specificity as the enzyme purified from serum. Rabbit serum PON3 is more efficient than rabbit PON1 in protecting low density lipoprotein from copper-induced oxidation. This is the first report that identifies a second PON enzyme in mammalian serum and the first to describe an enzymatic activity for PON3.     

Enhanced cholesteryl ester transfer protein activities and abnormalities of high density lipoproteins in familial hypercholesterolemia.

Cholesteryl ester transfer protein may play a role in the cholesteryl ester metabolism between high density lipoproteins (HDL) and apolipoprotein B-containing lipoproteins. To investigate relationship between HDL and cholesteryl ester transfer protein (CETP) activity in the development of atherosclerosis, the present study has focused on CETP activity in the patients with familial hypercholesterolemia (GH). HDL-C and HDL-C/apo A-I mass ratio in heterozygous FH were lower than those in normolipidemic controls. There was a 2-fold increase in total CETP activity in incubated FH serum compared with normolipidemic controls. Assays for CETP activity in the lipoprotein deficient serum (d greater than 1.215 g/ml) were carried out by measuring the transfer of radioactive cholesteryl ester from HDL (1.125 less than d less than 1.21 g/ml) to LDL (1.019 less than d less than 1.060 g/ml). CETP activities in heterozygous FH (79 +/- 4 nmol/ml/h) was significantly higher than those in normolipidemic controls (54 +/- 6 nmol/ml/h). The increased total cholesteryl ester transfer mainly results from increased CETP activity in the d greater than 1.215 g/ml, possibly reflecting an increase in CETP mass in serum. Increased CETP activity in the d greater than 1.215 g/ml was correlated positively with IDL-cholesterol/triglyceride mass ratio (r = 0.496, p less than 0.01), and negatively with HDL-cholesterol/apo A-I mass ratio (r = -0.334, p less than 0.05). These results indicate that the enhanced CETP activities may contribute to increase risk for developing atherosclerosis in FH by changing the distribution of cholesteryl ester in serum lipoproteins.     

Alterations of the plasma lipoproteins and apoproteins following cholesterol feeding in the rat.

The feeding of cholesterol to rats resulted in marked alterations in the type and distribution of the plasma lipoproteins and their apoproteins. The hyperlipoproteinemia was characterized by an increase in the d < 1.006 lipoproteins (B-VLDL and VLDL), an increase in the intermediate and low density lipoproteins (LDL), and the appearance of HDL(c). Associated with these lipoproteins was a prominence of the arginine-rich apoprotein. The high density lipoproteins (HDL) were decreased. A two-dimensional immunoelectrophoretic procedure was adapted to quantitate the changes in distribution of the arginine-rich apoprotein in the plasma and various ultracentrifugal fractions obtained from control and cholesterol-fed rats. In rats fed the cholesterol diet, the total plasma arginine-rich apoprotein increased from a control value of approximately 29 mg/dl to 47 mg/dl. The method of ultracentrifugation, however, was found to markedly alter the quantitative results. When the 60 Ti rotor was used at maximum speed to isolate the ultracentrifugal fractions, less than 50% of the total plasma arginine-rich apoprotein was associated with the lipoproteins in the d < 1.006 or the d 1.006-1.02, 1.02-1.063, or 1.063-1.21 ultracentrifugal fractions. By contrast, after limited ultracentrifugation with the 40 rotor, much less arginine-rich apoprotein was lost, with approximately 20% of the arginine-rich apoprotein in control rats and 10% in cholesterol-fed rats found in the d > 1.21 fraction. Significant alterations in the arginine-rich apoprotein quantitation notwithstanding, the observations of increased arginine-rich apoprotein in the B-VLDL, intermediate fraction, and HDL(c) following cholesterol feeding remained valid. However, precise quantitation awaits refinements in lipoprotein isolation techniques.     

Transfer of mouse anti-xenotropic virus neutralizing factor to human lipoproteins

We examined the migratory properties of the mouse serum protein responsible for specific neutralization of the endogenous mouse xenotropic type C virus and demonstrated that this neutralizing factor can be transferred from mouse lipoproteins to human and to nonreactive mouse lipoproteins. After acceptance of this activity by these lipoproteins, it could be back-transferred to human and to mouse lipoproteins. This result was compatible with equilibrium binding of the responsible proteins by several classes of lipoproteins. The best expression of the transferred activity occurred with human chylomicrons. Small quantities of neutralizing factor which exist in free dispersion in serum were also detected by mixing the p greater than 1.21 g/ml density fraction of mouse serum with human chylomicrons; antiviral activity was subsequently found associated with the human lipoproteins. This result reflects the potentiation of antiviral activity of the protein in a lipoprotein environment. These observations offer a useful approach for the eventual purification and characterization of the neutralizing factor.     

The levels of apolipoprotein-E in hypercholesterolemic rat serum.

The levels of apolipoprotein-C (apo-E) in serum and isolated liproproteins from diet-induced hypercholesterolemic, and to some extent hypertriglycerdemic rats were measured by electroimmunoassay. The hypocholesterolemia was accompanied by a mild hypertriglyceridemia. The apo-E was increased by 60% in the hypercholesterolemic serum with a 5- and 50-fold increase in very low density lipoproteins (VLDL) and low density lipoproteins (LDL) respectively. However, the proportion of apo-E in nascent VLDL isolated from the hepatic Golgi apparatus of hypercholesterolemic rats was significantly decreased. In control serum, 40--50% of the apo-E is found in the density greater than 1.21 g/ml fraction, although this is at least partially due to ultracentrifugation. The aproprotein is absent from the density greater than 1.21 g/ml fraction from hypercholesterolemic serum, suggesting that it is bound more firmly to the lipoprotein complex. It is concluded that the large increases in apo-E in the VLDL and LDL density ranges of serum from hypercholesterolemic rats may in part be accounted for by the utilization of apo-E normally found at higher densities.     

The sites of degradation of purified rat low density lipoprotein and high density lipoprotein in the rat

Low density lipoprotein and high density lipoprotein were isolated from rat serum by sequential ultracentrifugation in the density intervals 1.025-1.050 g/ml and 1.125-1.21 g/ml, respectively. The isolated lipoproteins were radioiodinated using ICl. Low density lipoprotein was further purified by concanavalin A affinity chromatography and concentrated by ultracentrifugation. 95% of the purified low density lipoprotein radioactivity was precipitable by tetramethylurea, while only 4% was associated with lipids. The radioiodinated high density lipoprotein was incubated for 1 h at 4 degrees C with unlabelled very low density lipoprotein, followed by reisolation by sequential ultracentrifugation. Only 3% of the radioactivity was associated with lipids and 90% was present on apolipoprotein A-I. The serum decay curves of labelled and subsequently purified rat low and high density lipoprotein, measured over a period of 28 h, clearly exhibited more than one component, in contrast to the monoexponential decay curves of iodinated human low density lipoprotein. The decay curves were not affected by the methods used to purify the LDL and HDL preparations. The catabolic sites of the labelled rat lipoproteins were analyzed in vivo using leupeptin-treated rats. In vivo treatment of rats with leupeptin did not affect the rate of disappearance from serum of intravenously injected labelled rat low density lipoprotein and high density lipoprotein. Leupeptin-dependent accumulation of radioiodine occurred almost exclusively in the liver after intravenous injection of iodinated low density lipoprotein, while both the liver and the kidneys showed leupeptin-dependent accumulation of radioactivity after injection of iodinated high density lipoprotein.     

Activity of paraoxonase 1 and lipid profile in healthy children

Paraoxonase 1 (PON1) seems to have a relevant role in detoxifying processes and in atherosclerosis. The aim of this study was to determine PON1 activity, the total antioxidant capacity, as well as entire lipid profile in children for screening of possible risk of atherosclerosis development. Serum PON1 arylesterase/paraoxonase activities were determined spectrophotometrically. The total antioxidant capacity of the serum was measured by TEAC method. Parameters of lipid profile were analyzed by routine laboratory methods. It has been shown that PON1 arylesterase/ paraoxonase activities were very similar to values found in adults. In children, no significant correlation between PON1 arylesterase activity and HDL was observed. PON1 paraoxonase activity correlated only with atherogenic index. PON1 arylesterase activity was significantly higher in girls than in boys. The antioxidant capacity was inversely related to the body mass index. In this study, PON1 activity was determined in healthy children aged 11 to 12 years and we found a similarity in PON1 activities of children and adults. Moreover, the results of our study support the hypothesis that higher body weight of children may contribute to a greater risk for development of atherosclerosis in which oxidative stress plays a role.     

Lipid Profile and Serum Characteristics of the Blind Subterranean Mole Rat,Spalax

Background

Spalax (blind subterranean mole rat), is a mammal adapted to live in fluctuating oxygen levels, and can survive severe hypoxia and hypercapnia. The adaptive evolution of Spalax to underground life resulted in structural and molecular-genetic differences comparing to above-ground mammals. These differences include higher myocardial maximal oxygen consumption, increased lung diffusion capacity, increased blood vessels density, and unique expression patterns of cancer and angiogenesis related genes such as heparanase, vascular endothelial growth factor, and P53.

Methodology/Principal Findings

Here we elucidate the main characteristics of Spalax lipid profile, as well as its main antioxidant and serum parameters. Compared to human, Spalax possesses lower total-cholesterol, low density lipoproteins (LDL) and triglycerides levels, and higher levels of high density lipoproteins (HDL). Apolipoprotein A-I and apolipoprotein B-100 were significantly lower in Spalax compared to human. Paraoxonase (PON) 1 arylesterase activity, was higher in Spalax compared to both human and mouse serum levels. Analysis of serum chemistry of Spalax revealed special features in this mammal.

Conclusions/Significance

Spalax possesses a unique lipid profile with high HDL and low LDL lipoproteins. The antioxidant serum content in the mole rat is higher than that of human and mouse. Serum C reactive protein (CRP) levels are significantly lower in Spalax compared to that of human or mouse, reflecting low levels of inflammation. These differences between Spalax, human and mouse are due to several factors including the intensive activity life-style that Spalax pursue underground, dietary components, and evolutionary genetic adaptations. Unfolding the genetic basis of these differences will probably result in unique treatments for a variety of human diseases such as dyslipedemias, inflammation and cancer.     

Evidence of the existence of a high-density lipoprotein transformation factor in pig and rabbit plasma

Experiments have been conducted to elucidate the incubation conditions necessary for the transformation of pig high-density lipoproteins (HDL) from particles resembling the HDL3 subfraction of human plasma into particles resembling human HDL2. All incubations were performed at 37 degrees C for 24 h in the presence of 0.002 M p-chloromercuriphenylsulphonic acid, a chemical inhibitor of lecithin-cholesterol acyltransferase. There was no transformation when pig HDL (1.09 less than d less than 1.21 g/ml) was incubated either in isolation or in the presence of bovine serum albumin. In addition, there was no transformation when the 1.25 g/ml infranatant of pig plasma was added to the incubation. In the presence of the 1.25 g/ml infranatant of rabbit plasma, by contrast, a significant proportion of the pig HDL was transformed into HDL2. The possibility that this result may have been the consequence of the lipid-transfer activity in rabbit plasma was excluded by the observation that a partially purified preparation of rabbit lipid-transfer protein was ineffective in promoting the HDL transformation. Experiments were also performed with the 1.09 g/ml infranatant of pig plasma. This fraction contained HDL and the plasma proteins but no other lipoprotein. In contrast to the absence of change in incubations of a mixture containing isolated pig HDL (1.09 less than d less than 1.21 g/ml) and the 1.25 g/ml infranatant of pig plasma, during incubation of the 1.09 g/ml infranatant there was an obvious formation of HDL2. It has been concluded that there exists in plasma an HDL-transformation factor which is distinct from both lecithin-cholesterol acyltransferase and the lipid-transfer protein. This factor was detectable in the 1.25 g/ml infranatant of rabbit plasma and in the 1.09 g/ml infranatant of pig plasma. As the factor was not detected in isolated pig HDL or in the 1.25 g/ml infranatant of pig plasma, the 1.21-1.25 g/ml fraction of plasma may be a useful starting point for further investigation of this factor.     

Apolipoprotein changes associated with the plasma lipid-regulating activity of gemfibrozil in cholesterol-fed rats

Gemfibrozil (Lopid) is a new plasma lipid-regulating drug that decreases very low and low density lipoprotein (VLD/LDL) and increases high density lipoprotein (HDL) concentrations in man. The present experiments tested the effects of gemfibrozil on plasma lipoproteins and apolipoproteins in rats fed high fat/high cholesterol diets. Compared to chow-fed rats, cholesterol feeding for 2 weeks (20% olive oil/2% cholesterol) produced the expected increases in VLDL and intermediate density lipoprotein (IDL) while lowering plasma HDL. This was documented by using three methods of lipoprotein isolation: sequential ultracentrifugation, density gradient ultracentrifugation, and agarose gel filtration. Gemfibrozil gavaged at 50 mg/kg per day for 2 weeks during cholesterol feeding prevented these changes such that lipoprotein patterns were similar to those in chow-fed animals. Whole plasma apoE and apoA-I concentrations were decreased and apoB increased due to cholesterol feeding as determined by electroimmunoassay, but again gemfibrozil treatment prevented these diet-induced alterations. Gradient polyacrylamide gel electrophoresis patterns of the total d less than 1.21 g/ml lipoprotein fractions reflected the changes in apolipoprotein concentrations and further demonstrated a greater increase of apoBl compared to apoBh in cholesterol-fed rats. Gemfibrozil lowered the concentration of both apoB variants and prevented the shift of apoE from HDL to lower density lipoproteins. Changes in the distribution of apoE were confirmed using agarose gel column chromatography followed by electroimmunoassay. These methods also revealed a shift of apoA-IV from HDL to the d greater than 1.21 g/ml, lipoprotein-free fraction with gemfibrozil treatment when blood was taken from fasted or postabsorptive animals. Since it was also noted that in chow-fed rats more apoA-IV was present in the d greater than 1.21 g/ml fraction in the postabsorptive or fed state compared to fasted animals, it could be postulated that the shift of apoA-IV into this fraction in gemfibrozil-treated rats is related to an accelerated clearance of chylomicrons. It is concluded that gemfibrozil largely prevents the accumulation of abnormal lipoproteins in this model of dyslipoproteinemia, and that apoE may play a critical role in this normalization process.     

VLDL triglycerides inhibit HDL-associated paraoxonase 1 (PON1) activity: in vitro and in vivo studies

We analyzed, for the first time, both in vitro and in vivo, the effect of very low density lipoprotein (VLDL), or of pure triglycerides, on high-density lipoprotein (HDL)-associated paraoxonase1 (PON1) catalytic activities. Incubation of serum or HDL from healthy subjects with VLDL (0-330 μg protein/mL) significantly decreased serum PON1 lactonase or arylesterase activities by up to 11% or 24%, and HDL-associated PON1 lactonase or arylesterase activities by up to 32% or 46%, respectively, in a VLDL dose-dependent manner. VLDL (0-660μg protein/mL) also inhibited recombinant PON1 (rePON1) lactonase or arylesterase activities by up to 20% or 42%, respectively. Similar inhibitory effect was noted upon rePON1 incubation with pure triglyceride emulsion. Bezafibrate therapy to three hypertriglyceridemic patients (400 mg/day, for one month) significantly decreased serum triglyceride concentration by 67%, and increased serum HDL cholesterol levels by 48%. PON1 arylesterase or paraoxonase activities in the patients' HDL fractions after drug therapy were significantly increased by 86-88%, as compared to PON1 activities before treatment. Similarly, HDL-PON1 protein levels significantly increased after bezafibrate therapy. Finally, bezafibrate therapy improved HDL biological activity, as HDL obtained after drug therapy showed increased ability to induce cholesterol efflux from J774A.1 macrophages, by 19%, as compared to HDL derived before therapy. We thus conclude that VLDL triglycerides inhibit PON1 catalytic activities, and bezafibrate therapy significantly improved HDL-PON1 catalytic and biological activities. ? 2012 International Union of Biochemistry and Molecular Biology, Inc.     

The very-high-density lipoprotein fraction of rabbit plasma is rich in tissue-derived cholesterol

When plasma from rabbits, which several weeks earlier had been infused with [3H]cholesterol, was subjected to equilibrium density gradient ultracentrifugation, the specific radioactivity of cholesterol in the very-high-density lipoprotein (VHDL) fraction (d 1.22-1.32 g/ml) was three to 8-fold greater (mean, 5.5-fold; P less than 0.001) than that in high-density lipoproteins (HDL; d 1.06-1.21 g/ml). On size exclusion chromatography of plasma, no increase in specific radioactivity was seen in particles smaller than HDL. These findings suggest that those apolipoprotein-lipid complexes that dissociate from HDL during ultracentrifugation to form the VHDL fraction contain proportionately more tissue-derived cholesterol than do those that are more tightly bound to HDL.