Human Plasma Phospholipid Transfer Protein Research Articles

Plasma Phospholipid Transfer Protein Promotes Platelet Aggregation.

It remains unclear whether plasma phospholipid transfer protein (PLTP) is involved in hyper-coagulation or hypo-coagulation. This study investigated the direct effect of PLTP on platelet aggregation and the underlying mechanism. Washed platelets from humans or mice and mouse platelet-rich plasma and human recombinant PLTP were isolated. PLTP is present in human platelets. We assessed adenosine diphosphate (ADP)-, collagen- and thrombin-induced platelet aggregation, phosphatidylserine externalization and photothrombosis-induced cerebral infarction in mice. PLTP over-expression increased platelet aggregation, while PLTP deficiency had the opposing reaction. Human recombinant PLTP increased both mouse and human platelet aggregation in a dose-dependent manner. Phosphatidylserine externalization provides a water/lipid surface for the interaction of coagulation factors, which accelerates thrombosis. Compared with wild-type controls, platelets from PLTP transgenic mice had significantly more phosphatidylserine on the exterior surface of the plasma membrane, whereas platelets from PLTP-deficient mice had significantly less phosphatidylserine on the surface, thus PLTP influences fibrinogen binding on the plasma membrane. Moreover, recombinant PLTP together with ADP significantly increased phosphatidylserine exposure on the plasma membrane of PLTP-deficient platelets, thereby increasing fibrinogen binding. PLTP over-expression significantly accelerated the incidence of photothrombosis-induced infarction in mice, whereas PLTP deficiency significantly reduced the frequency of infarction. We concluded that PLTP promotes phosphatidylserine externalization at the plasma membrane of platelets and accelerates ADP- or collagen-induced platelet aggregation. This effect plays an important role in the initiation of thrombin generation and platelet aggregation under sheer stress conditions. Thus, PLTP is involved in hyper-coagulation. Therefore, PLTP inhibition could be a novel approach for countering thrombosis.

Recombinant human plasma phospholipid transfer protein (PLTP) to prevent bacterial growth and to treat sepsis

Although plasma phospholipid transfer protein (PLTP) has been mainly studied in the context of atherosclerosis, it shares homology with proteins involved in innate immunity. Here, we produced active recombinant human PLTP (rhPLTP) in the milk of new lines of transgenic rabbits. We successfully used rhPLTP as an exogenous therapeutic protein to treat endotoxemia and sepsis. In mouse models with injections of purified lipopolysaccharides or with polymicrobial infection, we demonstrated that rhPLTP prevented bacterial growth and detoxified LPS. In further support of the antimicrobial effect of PLTP, PLTP-knocked out mice were found to be less able than wild-type mice to fight against sepsis. To our knowledge, the production of rhPLTP to counter infection and to reduce endotoxemia and its harmful consequences is reported here for the first time. This paves the way for a novel strategy to satisfy long-felt, but unmet needs to prevent and treat sepsis.

Phospholipid Transfer Protein Is Differentially Expressed in Human Arterial and Venous Placental Endothelial Cells and Enhances Cholesterol Efflux to Fetal HDL

Phospholipid (PL) transfer protein (PLTP) plays a crucial role in high-density lipoprotein (HDL) metabolism. In the fetal circulation, HDL particles are the main cholesterol carriers and are involved in maternal-fetal cholesterol transfer across human placental endothelial cells (HPEC). The aim was to investigate local function(s) of PLTP at the fetoplacental endothelium. Because HPEC display morphological and functional diversity when isolated from arteries or veins, we hypothesized that PLTP activity may differ between arterial and venous HPEC. We determined PLTP mRNA and activity levels from isolated HPEC and investigated PLTP-mediated remodeling of fetal HDL particles and their capacity in mediating cholesterol efflux from HPEC. Incubation of fetal HDL with active human plasma PLTP resulted in increased particle size (12.6 vs. 13.2 nm, P < 0.05), with a concomitant increase (3.5-fold) in pre-β-mobile HDL particles. Arterial HPEC showed higher Pltp expression levels and secreted PL transfer activity (1.8-fold, P < 0.001) than venous HPEC. In contrast to adult HDL(3), [(3)H]cholesterol efflux to fetal HDL was 21% higher (P < 0.05) from arterial than from venous HPEC. PLTP-facilitated particle conversion increased the cholesterol efflux capacity of fetal HDL to similar extents (55 and 48%, P < 0.001) from arterial and venous HPEC, respectively. PLTP mediates PL transfer and participates in reverse cholesterol transport pathways at the fetoplacental barrier. Enhanced cellular cholesterol efflux from HPEC to fetal HDL remodeled by PLTP supports the idea of a local atheroprotective role of PLTP in the placental vasculature.

Worsening of Diet-Induced Atherosclerosis in a New Model of Transgenic Rabbit Expressing the Human Plasma Phospholipid Transfer Protein

Plasma phospholipid transfer protein (PLTP) is involved in intravascular lipoprotein metabolism. PLTP is known to act through 2 main mechanisms: by remodeling high-density lipoproteins (HDL) and by increasing apolipoprotein (apo) B-containing lipoproteins. The aim of this study was to generate a new model of human PLTP transgenic (HuPLTPTg) rabbit and to determine whether PLTP expression modulates atherosclerosis in this species that, unlike humans and mice, displays naturally very low PLTP activity. In HuPLTPTg rabbits, the human PLTP cDNA was placed under the control of the human eF1-α gene promoter, resulting in a widespread tissue expression pattern and in increased plasma PLTP. The HuPLTPTg rabbits showed a significant increase in the cholesterol content of the plasma apoB-containing lipoprotein fractions, with a more severe trait when animals were fed a cholesterol-rich diet. In contrast, HDL cholesterol level was not modified in HuPLTPTg rabbits. Formation of aortic fatty streaks was increased in hypercholesterolemic HuPLTPTg animals as compared with nontransgenic littermates. Human PLTP expression in HuPLTPTg rabbit worsens atherosclerosis as a result of increased levels of atherogenic apoB-containing lipoproteins but not of alterations in their antioxidative protection or in cholesterol content of plasma HDL.

Human plasma phospholipid transfer protein specific activity is correlated with HDL size: Implications for lipoprotein physiology

To gain further insights into the relationship between plasma phospholipid transfer protein (PLTP) and lipoprotein particles, PLTP mass and phospholipid transfer activity were measured, and their associations with the level and size of lipoprotein particles examined in 39 healthy adult subjects. No bivariate correlation was observed between PLTP activity and mass. PLTP activity was positively associated with cholesterol, triglyceride, apo B and VLDL particle level ( r s = 0.40–0.56, p ≤ 0.01) while PLTP mass was positively associated with HDL-C, large HDL particles, and mean LDL and HDL particle sizes ( r s = 0.44–0.52, p < 0.01). Importantly, plasma PLTP specific activity (SA) was significantly associated with specific lipoprotein classes, positively with VLDL, IDL, and small LDL particles ( r s = 0.42–0.62, p ≤ 0.01) and inversely with large LDL, large HDL, and mean LDL and HDL particle size ( r s = − 0.42 to − 0.70, p ≤ 0.01). After controlling for triglyceride levels, the correlation between PLTP mass or SA and HDL size remained significant. In linear models, HDL size explained 45% of the variability of plasma PLTP SA while triglyceride explained 34% of the PLTP activity. Thus, in healthy adults a significant relationship exists between HDL size and plasma PLTP SA ( r s = − 0.70), implying that HDL particle size may modulate PLTP SA in the vascular compartment.

Plasma phospholipid transfer activity is essential for increased atherogenesis in PLTP transgenic mice: a mutation-inactivation study

Plasma phospholipid transfer protein (PLTP) interacts with HDL particles and facilitates the transfer of phospholipids from triglyceride (TG)-rich lipoproteins to HDL. Overexpressing human PLTP in mice increases the susceptibility to atherosclerosis. In human plasma, high-active and low-active forms of PLTP exist. To elucidate the contribution of phospholipid transfer activity to changes in lipoprotein metabolism and atherogenesis, we developed mice expressing mutant PLTP, still able to associate with HDL but lacking phospholipid transfer activity. In mice heterozygous for the LDL receptor, effects of the mutant and normal human PLTP transgene (mutPLTP tg and PLTP tg, respectively) were compared. In PLTP tg mice, plasma PLTP activity was increased 2.9-fold, resulting in markedly reduced HDL lipid levels. In contrast, in mutPLTP tg mice, lipid levels were not different from controls. Furthermore, hepatic VLDL-TG secretion was stimulated in PLTP tg mice, but not in mutPLTP tg mice. When mice were fed a cholesterol-enriched diet, atherosclerotic lesion size in PLTP tg mice was increased more than 2-fold compared with control mice, whereas in mutPLTP tg mice, there was no change. Our findings demonstrate that PLTP transfer activity is essential for the development of atherosclerosis in PLTP transgenic mice, identifying PLTP activity as a possible target to prevent atherogenesis, independent of plasma PLTP concentration.

Phospholipid transfer protein deficiency ameliorates diet-induced hypercholesterolemia and inflammation in mice

Phospholipid transfer protein (PLTP) facilitates the transfer of phospholipids from triglyceride-rich lipoproteins into HDL. PLTP has been shown to be an important factor in lipoprotein metabolism and atherogenesis. Here, we report that chronic high-fat, high-cholesterol diet feeding markedly increased plasma cholesterol levels in C57BL/6 mice. PLTP deficiency attenuated diet-induced hypercholesterolemia by dramatically reducing apolipoprotein E-rich lipoproteins (-88%) and, to a lesser extent, LDL (-40%) and HDL (-35%). Increased biliary cholesterol secretion, indicated by increased hepatic ABCG5/ABCG8 gene expression, and decreased intestinal cholesterol absorption may contribute to the lower plasma cholesterol in PLTP-deficient mice. The expression of proinflammatory genes (intercellular adhesion molecule-1 and vascular cell adhesion molecule-1) is reduced in aorta of PLTP knockout mice compared with wild-type mice fed either a chow or a high-cholesterol diet. Furthermore, plasma interleukin-6 levels are significantly lower in PLTP-deficient mice, indicating reduced systemic inflammation. These data suggest that PLTP appears to play a proatherogenic role in diet-induced hyperlipidemic mice.

Atherogenic, enlarged, and dysfunctional HDL in human PLTP/apoA-I double transgenic mice

In low density lipoprotein receptor (LDLR)-deficient mice, overexpression of human plasma phospholipid transfer protein (PLTP) results in increased atherosclerosis. PLTP strongly decreases HDL levels and might alter the antiatherogenic properties of HDL particles. To study the potential interaction between human PLTP and apolipoprotein A-I (apoA-I), double transgenic animals (hPLTPtg/hApoAItg) were compared with hApoAItg mice. PLTP activity was increased 4.5-fold. Plasma total cholesterol and phospholipid were decreased. Average HDL size (analyzed by gel filtration) increased strongly, hPLTPtg/hApoAItg mice having very large, LDL-sized, HDL particles. Also, after density gradient ultracentrifugation, a substantial part of the apoA-I-containing lipoproteins in hPLTPtg/hApoAItg mice was found in the LDL density range. In cholesterol efflux studies from macrophages, HDL isolated from hPLTPtg/hApoAItg mice was less efficient than HDL isolated from hApoAItg mice. Furthermore, it was found that the largest subfraction of the HDL particles present in hPLTPtg/hApoAItg mice was markedly inferior as a cholesterol acceptor, as no labeled cholesterol was transferred to this fraction. In an LDLR-deficient background, the human PLTP-expressing mouse line showed a 2.2-fold increased atherosclerotic lesion area. These data demonstrate that the action of human PLTP in the presence of human apoA-I results in the formation of a dysfunctional HDL subfraction, which is less efficient in the uptake of cholesterol from cholesterol-laden macrophages.

Absence of endogenous phospholipid transfer protein impairs ABCA1-dependent efflux of cholesterol from macrophage foam cells

In vitro experiments have demonstrated that exogenous phospholipid transfer protein (PLTP), i.e. purified PLTP added to macrophage cultures, influences ABCA1-mediated cholesterol efflux from macrophages to HDL. To investigate whether PLTP produced by the macrophages (i.e., endogenous PLTP) is also part of this process, we used peritoneal macrophages derived from PLTP-knockout (KO) and wild-type (WT) mice. The macrophages were transformed to foam cells by cholesterol loading, and this resulted in the upregulation of ABCA1. Such macrophage foam cells from PLTP-KO mice released less cholesterol to lipid-free apolipoprotein A-I (apoA-I) and to HDL than did the corresponding WT foam cells. Also, when plasma from either WT or PLTP-KO mice was used as an acceptor, cholesterol efflux from PLTP-KO foam cells was less efficient than that from WT foam cells. After cAMP treatment, which upregulated the expression of ABCA1, cholesterol efflux from PLTP-KO foam cells to apoA-I increased markedly and reached a level similar to that observed in cAMP-treated WT foam cells, restoring the decreased cholesterol efflux associated with PLTP deficiency. These results indicate that endogenous PLTP produced by macrophages contributes to the optimal function of the ABCA1-mediated cholesterol efflux-promoting machinery in these cells. Whether macrophage PLTP acts at the plasma membrane or intracellularly or shuttles between these compartments needs further study.

Sex differences in atherosclerosis in mice with elevated phospholipid transfer protein activity are related to decreased plasma high density lipoproteins and not to increased production of triglycerides

Plasma phospholipid transfer protein (PLTP) has atherogenic properties in genetically modified mice. PLTP stimulates hepatic triglyceride secretion and reduces plasma levels of high density lipoproteins (HDL). The present study was performed to relate the increased atherosclerosis in PLTP transgenic mice to one of these atherogenic effects. A humanized mouse model was used which had decreased LDL receptor expression and was transgenic for human cholesterylester transfer protein (CETP) in order to obtain a better resemblance to the plasma lipoprotein profile present in humans. It is well known that female mice are more susceptible to atherosclerosis than male mice. Therefore, we compared male and female mice expressing human PLTP. The animals were fed an atherogenic diet and the effects on plasma lipids and lipoproteins, triglyceride secretion and the development of atherosclerosis were measured. The development of atherosclerosis was sex-dependent. This effect was stronger in PLTP transgenic mice, while PLTP activity levels were virtually identical. Also, the rates of hepatic secretion of triglycerides were similar. In contrast, plasma levels of HDL were about 2-fold lower in female mice than in male mice after feeding an atherogenic diet. We conclude that increased atherosclerosis caused by overexpression of PLTP is related to a decrease in HDL, rather than to elevated hepatic secretion of triglycerides.

N-Glycosylation is Required for Secretion-Competent Human Plasma Phospholipid Transfer Protein

Human plasma phospholipid transfer protein (PLTP) contains six potential N-glycosylation sites (Asn-X-Ser). To study the role of these sites on PLTP structure and function, seven variants in which asparagine (N) residues were converted to glycine (G) were prepared by site-directed mutagenesis. These were N(47)G, N(77)G, N(100)G, N(126)G, N(228)G, N(381)G and N(47, 77, 100, 126, 228, 381)G (N(null)G). These variants and wild-type (WT) PLTP were expressed in COS-7 cells. Intracellular and secreted PLTP mass was analyzed by Western blots and quantitative enzyme-linked immunosorbent assay; PLTP activities in cellular lysates and media were based on the transfer of [(3)H]dipalmitoylphosphatidylcholine from phospholipid single bilayer vesicles to HDL. N(null)G was not detected intracellularly. N(381)G was similar to WT PLTP with respect to specific activity and secretion efficiency. The specific activities of N(47)G, N(77)G, N(100)G, N(126)G, N(228)G and N(381)G were similar in cell lysate (range = 67-90% WT) and medium (range = 65-77% WT). Intracellular masses of these PLTP variants were similar to that of WT (Mean = 103% WT); mean secreted mass was 88% WT. These results suggest that secretion-competent PLTP requires glycosylation but that no single glycosylation site is required.

Fenofibrate reverses the decline in HDL cholesterol in mice overexpressing human phospholipid transfer protein

In humans, fibrates are used to treat dyslipidemia, because these drugs lower plasma triglycerides and raise HDL cholesterol. Treatment with fibrates lowers plasma phospholipid transfer protein (PLTP) activity in humans, but increases PLTP activity in mice, without a consistent effect on HDL-cholesterol concentration. Earlier, we found that PLTP overexpression in transgenic mice results in decreased plasma HDL levels and increased diet-induced atherosclerosis. So it seems that the interplay between fibrates, PLTP and HDL is different in mice and man, which may be important for atherosclerosis development. In the present study, we measured the effects of fibrates on PLTP expression in cultured human hepatocytes and effects of fibrate treatment on human PLTP expression, plasma PLTP activity and HDL levels in human PLTP transgenic mice. Fibrate treatment did not influence PLTP mRNA levels in human hepatocytes. Hepatic human PLTP mRNA levels and PLTP activity were both moderately elevated by fenofibrate treatment in human PLTP transgenic mice. In wild-type mice, however, feeding fenofibrate resulted in a strong induction of PLTP mRNA in the liver and a more than 4-fold increase of plasma PLTP activity. Plasma triglycerides were reduced in all mice by 48% or more by fenofibrate treatment. HDL-cholesterol concentrations were substantially increased by fenofibrate in PLTP overexpressing mice (+72%), but unaffected in wild-type mice. We conclude that fenofibrate treatment reverses the HDL-lowering effect of PLTP overexpression in human PLTP transgenic mice.

Remodeling of Human Plasma Lipoproteins by Detergent Perturbation

Detergent perturbation, the treatment of total human plasma lipoproteins (TLP) with sodium cholate and its subsequent removal, has been used to study lipoprotein dynamics and stability. At physiological TLP concentrations, detergent perturbation converts low-density lipoproteins (LDL) and high-density lipoproteins (HDL) to higher-particle weight species with the concomitant release of apo A-I but not apo A-II as a lipid-poor species. Detergent perturbation of isolated HDL also releases lipid-poor apo A-I and forms larger HDL species, whereas detergent perturbation of an isolated LDL has no effect on its size. A model is presented in which detergent perturbation induces transfer of PC from metastable HDL and LDL to mixed micelles with sodium cholate. The remaining LDL and HDL are unstable because of the loss of their surface components, phospholipid and/or apo A-I, and fuse to give larger LDL and HDL particles. These effects on HDL, i.e., PC transfer, apo A-I dissociation, and particle fusion, emulate the activity of human plasma phospholipid transfer protein. Thus, detergent perturbation is a new and potentially powerful method for determining lipoprotein stability, studying the mechanisms for remodeling of plasma lipoproteins, and preparing new forms of HDL and LDL with unique interactions with lipoprotein transporters and receptors.

Human plasma phospholipid transfer protein activity is decreased by acute hyperglycaemia: studies without and with hyperinsulinaemia in Type 1 diabetes mellitus

Little is known about the regulation of phospholipid transfer protein (PLTP), that plays a key role in lipoprotein metabolism. PLTP secretion may be up-regulated by glucose in vitro, whereas plasma PLTP activity is decreased by exogenous hyperinsulinaemia and glucose-induced hyperinsulinaemia in vivo. In the present study, we evaluated the separate effects of hyperglycaemia and hyperinsulinaemia in C-peptide-negative Type 1 diabetic patients. The protocol was carried out in 16 patients (eight females). In each individual, plasma PLTP mass and activity (measured by enzyme-linked immuno-sorbent assay and liposome-high density lipoprotein system, respectively) as well as plasma cholesteryl ester transfer protein (CETP) activity, lipids and apolipoprotein levels were determined at the end of four different glucose clamps, each lasting 210 min: standard insulin (30 mU/kg/h) and standard glucose (glucose 5.0 mmol/l) (SI-SG), standard insulin and high glucose (glucose 12 mmol/l) (SI-HG), high insulin (150 mU/kg/h) and standard glucose (HI-SG), and high insulin and high glucose (HI-HG). Plasma lipids and (apo)lipoproteins, measured at the end of the SI-HG, HI-SG and HI-HG clamps, were not significantly different compared with the levels obtained at the end of the SI-SG clamp. Median plasma PLTP mass and activity at the end of the SI-SG clamp were 12.8 mg/l and 13.2 micromol/ml/h, respectively. Median plasma PLTP mass decreased by 9.1% at the end of the HI-HG clamp (P < 0.01), whereas the changes at the end of the SI-HG and HI-SG clamps were not significant. Median plasma PLTP activity decreased by 5.7, 4.6 and 8.6% at the end of the SI-HG, HI-SG and HI-HG clamps, respectively (all P < 0.05). Median plasma CETP activity was 177 nmol/ml/h at the end of the SI-SG clamp, and decreased by 4.9% (P < 0.05) and by 8.3% (P < 0.05) at the end of the HI-SG and the HI-HG clamps, respectively. Plasma CETP activity did not change significantly at the end of the SI-HG clamp. The present study demonstrates that plasma PLTP activity is independently decreased by acute hyperglycaemia and hyperinsulinaemia in humans in vivo. These data do not support a direct role of short-term hyperglycaemia in up-regulating plasma PLTP levels.

Determination of human plasma phospholipid transfer protein mass and activity

Plasma phospholipid transfer protein (PLTP) plays an important role in lipoprotein metabolism. PLTP is an 80-kDa glycoprotein that is expressed/secreted by a wide variety of tissues including lung, liver, adipose tissue, brain, and muscle. PLTP mediates a net transfer of phospholipids between vesicles and plasma HDLs. It also generates from small HDL particles large fused HDL particles with a concomitant formation of small lipid-poor apolipoprotein (apo) A-I-containing particles which are thought to act as the primary acceptors of cell-derived cholesterol from peripheral tissue macrophages. Another important function of PLTP is connected to lipolysis. Its role in the transfer of surface remnants from triglyceride-rich particles, very-low-density lipoproteins, and chylomicrons, to HDL is of importance for the maintenance of HDL levels. Recent observations from our laboratory have demonstrated that in circulation two forms of PLTP are present, one catalytically active (high-activity form, HA-PLTP) and the other a low-activity form (LA-PLTP). In view of the likely relevancy of PLTP in human health and disease, reliable and accurate methods for measuring plasma/serum PLTP activity and concentration are required. In this chapter, two radiometric PLTP activity assays are described: (i) exogenous, lipoprotein-independent phospholipid transfer assay and (ii) endogenous, lipoprotein-dependent phospholipid transfer assay. In addition, an ELISA method for quantitation of serum/plasma total PLTP mass as well as HA-PLTP and LA-PLTP mass is reported in detail.

Low-Density Lipoprotein Inhibits Secretion of Phospholipid Transfer Protein in Human Trophoblastic BeWo Cells

Human plasma phospholipid transfer protein (PLTP) plays an important role in lipoprotein metabolism. In this study, we investigated the effects of lipoproteins on the secretion of PLTP in cultured BeWo choriocarcinoma cells. Low-density lipoproteins (LDLs) decreased PLTP secretion in a dose- and time-dependent manner, whereas very low density lipoproteins and high-density lipoproteins (HDLs) had little effect. LDL suppression of PLTP secretion was not altered by the inhibition of both LDL receptor and LDL receptor-related protein with receptor-associated protein. Mitogen-activated protein kinase (MAPK) kinase (MEK) inhibitor, U0126, could abolish the LDL-mediated inhibition of PLTP secretion. Furthermore, LDL, but not HDL, could stimulate the expression of MAPK phosphatase-1 (MKP-1) in BeWo cells that resulted in the inactivation of p44/p42 extracellular signal-regulated kinase (ERK) 1 and 2, the family members of MAPKs. These results support the conclusion that LDL-mediated suppression of PLTP secretion in BeWo cells is through a LDL receptor-independent MAPK signaling pathway.

Elevation of plasma phospholipid transfer protein increases the risk of atherosclerosis despite lower apolipoprotein B-containing lipoproteins

Plasma phospholipid transfer protein (PLTP) transfers phospholipids between lipoproteins and mediates HDL conversion. PLTP-overexpressing mice have increased atherosclerosis. However, mice do not express cholesteryl ester transfer protein (CETP), which is involved in the same metabolic pathways as PLTP. Therefore, we studied atherosclerosis in heterozygous LDL receptor-deficient (LDLR(+/-)) mice expressing both human CETP and human PLTP. We used two transgenic lines with moderately and highly elevated plasma PLTP activity. In LDLR(+/-)/huCETPtg mice, cholesterol is present in both LDL and HDL. Both are decreased in LDLR(+/-)/huCETPtg/huPLTPtg mice (>50%). An atherogenic diet resulted in high levels of VLDL+LDL cholesterol. PLTP expression caused a strong PLTP dose-dependent decrease in VLDL and LDL cholesterol (-26% and -69%) and a decrease in HDL cholesterol (-70%). Surprisingly, atherosclerosis was increased in the two transgenic lines with moderately and highly elevated plasma PLTP activity (1.9-fold and 4.4-fold, respectively), indicating that the adverse effect of the reduction in plasma HDL outweighs the beneficial effect of the reduction in apolipoprotein B (apoB)-containing lipoproteins. The activities of the antiatherogenic enzymes paraoxonase and platelet-activating factor acetyl hydrolase were both PLTP dose-dependently reduced ( approximately -33% and -65%, respectively). We conclude that expression of PLTP in this animal model results in increased atherosclerosis in spite of reduced apoB-containing lipoproteins, by reduction of HDL and of HDL-associated antioxidant enzyme activities.

W11.282 Development of a new elisa to quantitative the active and low-active forms of human plasma phospholipid transfer protein (PLTP)

W11.282 Development of a new elisa to quantitative the active and low-active forms of human plasma phospholipid transfer protein (PLTP)

W11.282 Development of a new elisa to quantitative the active and low-active forms of human plasma phospholipid transfer protein (PLTP)

W11.282 Development of a new elisa to quantitative the active and low-active forms of human plasma phospholipid transfer protein (PLTP)

Quantitation of the active and low-active forms of human plasma phospholipid transfer protein by ELISA

Human plasma contains two forms of phospholipid transfer protein (PLTP), one catalytically active [high-activity PLTP (HA-PLTP)] and the other a low-activity (LA-PLTP) form. We present here a PLTP ELISA that allows not only for accurate measurement of PLTP concentration in plasma but also of the distribution of both LA- and HA-PLTP. To achieve similar immunoreactivity of the two PLTP forms, a denaturing sample pretreatment with 0.5% SDS was required. Distribution of LA- and HA-PLTP in plasma was assessed using size-exclusion chromatography, Heparin-Sepharose chromatography, anti-PLTP immunoaffinity chromatography, and dextran sulfate-CaCl2 precipitation. All four methods demonstrated that approximately 60% of plasma PLTP represents LA-PLTP and 40% represents HA-PLTP. According to the modified ELISA, the total serum PLTP concentration in a random Finnish population sample (n = 80) was 5.81 +/- 1.33 mg/l (mean +/- SD) (range, 2.78-10.06 mg/l) and the mean activity was 5.84 +/- 1.39 micromol/ml/h (range, 3.21-11.15 micromol/ml/h). To quantitate both forms of PLTP in sera from this sample, we combined dextran sulfate-CaCl2 precipitation with the modified PLTP ELISA. The HA-PLTP mass (mean, 1.87 +/- 0.85 mg/l) correlated significantly with serum PLTP activity, whereas that of LA-PLTP (mean, 3.94 +/- 1.4 mg/l) showed no correlation with phospholipid transfer activity.