Label-free electrochemical homogeneous detection of the depression marker human apolipoprotein A4 based on proximity hybridization triggered rolling circle amplification.

In this paper, we developed a label-free homogeneous electrochemical sensor for detection of apolipoprotein A4 based on proximity hybridization triggered rolling circle amplification induced G-quadruplex formation. The presence of apolipoprotein A4 promoted the formation of a proximate complex via the proximity hybridization of the aptamer DNA strands, which unfolded the molecular beacon, the stem part of molecular beacon as a primer to initiate the RCA process. Thus, with the electrochemical indicator hemin selectively intercalated into the multiple G-quadruplexes, a significant electrochemical signal drop is observed, which is dependent on the concentration of the target apolipoprotein A4. Thus, using this "signal-off" mode, label-free homogeneous electrochemical strategy for sensitive apolipoprotein A4 assay with a wide range from 1 pg mL-1 to 100 ng mL-1, with a low detection limit of 0.51 pg mL-1. And it rendered satisfactory analytical performance for the determination of apolipoprotein A4 in serum samples. Furthermore, this method also exhibits additional advantages of simplicity and low cost, since both expensive labeling and sophisticated probe immobilization processes are avoided. The satisfactory results indicated that the proposed sensor had promising potential in the clinical diagnosis of depression.

Anti-apolipoprotein A-1 autoantibodies correlate with disease activity in systemic lupus erythematosus.

OBJECTIVES: Apolipoprotein A-1 (ApoA-1) is a protein fraction of the high-density lipoproteins with anti-inflammatory and antioxidant properties that play a major role in reverse cholesterol transport. The presence of anti-ApoA-1 IgG has been reported in SLE to be variably associated with disease activity or cardiovascular events (CVEs). We assessed the clinical performance of anti-ApoA-1 IgG and of antibodies directed against its immunodominant F3L1 peptide (F3L1 IgG) in a well-characterized Swiss SLE cohort study. METHODS: A total of 354 biological samples and interviews from 176 individuals were studied. SLEDAI, clinical characteristics, anamnestic CVEs and therapy details were recorded. Sera were tested for the presence of anti-ApoA-1 IgG, anti-F3L1 IgG, anti-dsDNA IgG and aPL. RESULTS: Anti-ApoA-1 and anti-F3L1 IgG positivity was associated with higher SLEDAI, mostly due to concomitant positivity of dsDNA IgG and low complement. Variations in time of anti-ApoA-1 IgG correlated positively with variations of anti-dsDNA IgG and inversely to variations of C3 levels. No cross-reactivity was found between anti-ApoA-1 and anti-dsDNA IgG. Positivity for anti-Apo-A1 IgG was more frequent in individuals receiving 10 mg/day or more of prednisone. We did not find any significant association between anti-ApoA-1 IgG positivity and CVEs. CONCLUSION: Anti-ApoA-1 and anti-F3L1 IgG in SLE correlate strongly with laboratory markers of activity, particularly with the presence and titre of dsDNA IgG. These results confirm and extend previous findings and support the use of anti-ApoA1 IgG in the clinical setting. Their role in CVEs deserves further investigation.

Characterization, expression and antibacterial properties of apolipoproteins A from carp (Cyprinus carpio L.) seminal plasma.

Apolipoproteins A are multifunctional proteins that, in addition to contributing to lipid metabolism and transport, are associated with the innate immune system in fish. Using a three step isolation procedure consisting of affinity chromatography on Blue-Sepharose, delipidation and reverse phase HPLC we isolated apolipoproteins from carp seminal plasma and identified them as ApoA-I and Apo-14 kDa. Moreover, we provided the full-length cDNA sequence of ApoA-I encoding 257 amino acids including a 18 amino acid signal peptide and a 4 amino acid propeptide. Apolipoproteins corresponded to the most abundant proteins in carp seminal plasma. Both ApoA-I and Apo-14 kDa were represented by several proteoforms that differ both in molecular mass and isoelectric point. The proteoforms of ApoA-I characteristic for seminal plasma were distinguished from those of blood. Carp seminal plasma ApoA-I and Apo-14 kDa showed a high immunologic similarity to their counterparts in carp blood and seminal plasma of other Cyprinid species. The mRNA expression analysis and immunohistochemical study suggest synthesis and secretion of ApoA-I and Apo-14 kDa in the fish reproductive tract and suggest a role in spermatogenesis and the stabilization of sperm membrane. Moreover, ApoA-I displayed bactericidal activity against Escherichia coli and bacteriostatic activity against Aeromonas hydrophila which suggests that ApoA-I is associated with innate immune system of the fish reproductive tract.

Plasma anti-α-galactoside antibody binds to serine- and threonine-rich peptide sequence of apo(a) subunit in Lp(a).

Lipoprotein(a) immune complexes [Lp(a) IC] of varying particle density obtained by ultracentrifugation of plasma from normal healthy donors were markedly dominated by IgG. Lp(a) and immunoglobulins were liberated from plasma Lp(a) IC by treatment with melibiose, a sugar specific for circulating anti-α-galactoside antibody (anti-Gal). Upon incubation with plasma lipoprotein fraction anti-Gal but not the α-glucoside-specific antibody from human plasma formed de novo IC with Lp(a). Binding of Lp(a) sugar-reversibly enhanced the fluorescence of FITC-labeled anti-Gal as did binding of α-galactoside-containing glycoproteins. This effect apparently due to conformational shift in the Fc region of the antibody was also produced by apo(a) subunit separated from Lp(a) and de-O-glycosylated apo(a) but not by any other plasma lipoproteins or by Lp(a) pre-incubated with the O-glycan-specific lectin jacalin. O-Glycans and their terminal sialic acid moieties in apo(a) of circulating Lp(a)-anti-Gal IC, in contrast to those in pure Lp(a), were inaccessible to jacalin and anion exchange resin, respectively. Unlike other plasma lipoproteins, Lp(a) inhibited Griffonia simplicifolia isolectin B4 which also accommodates serine- and threonine-rich peptide sequence (STPS) as surrogate ligand to α-galactosides at its binding site. Results suggest that anti-Gal recognizes STPS in the O-glycan-rich regions of apo(a) subunit in Lp(a) which contains no α-linked galactose.

Human plasma anti-α-galactoside antibody forms immune complex with autologous lipoprotein(a).

Anti-α-galactoside antibody (anti-Gal) from human plasma that bound to α-galactoside-bearing guar galactomannan gel and was eluted with specific sugar (affinity-purified anti-Gal ; APAG) invariably contained apo(a) and apo B subunits in a proportion close to that in plasma lipoprotein(a) [Lp(a)]. Since LDL does not contain apo(a), result suggested Lp(a) as a component of APAG. Lp(a) in APAG was complexed with anti-Gal since plate-coated anti-apo(a) captured Lp(a) along with the antibody. Association of Lp(a) with anti-Gal in APAG was considerably lower in presence of anti-Gal-specific sugar, suggesting that Lp(a) occupied the sugar-binding site of anti-Gal. Content of Lp(a)-bound anti-Gal in APAG, though a minor fraction of total antibody, increased steadily with total Lp(a) content of plasma. Further, Lp(a) released from immune complex-rich fraction of plasma by anti-Gal- specific sugar was proportional to total plasma Lp(a). Anti-Gal titre decreased with increasing Lp(a) concentration among 114 plasma samples. Results indicate the potential of anti-Gal molecules with its binding site partially occupied by Lp(a) molecule(s) to a) use the remaining binding site(s) to recognize other macromolecules or cells and b) transport Lp(a) across Fc receptor-bearing cells.

Immunohistochemical localization of apolipoprotein A-IV in human kidney tissue.

BACKGROUND: Apolipoprotein A-IV (ApoA-IV) is a 46 kD glycoprotein thought to protect against atherosclerosis. It is synthesized primarily in epithelial cells of the small intestine. Elevated plasma concentrations of ApoA-IV in patients with chronic kidney disease suggest that the human kidney is involved in ApoA-IV metabolism. METHODS: To investigate whether the human kidney directly metabolizes ApoA-IV and which kidney tissue compartment is involved therein, ApoA-IV was localized by immunohistochemistry in 28 healthy kidney tissue samples obtained from patients undergoing nephrectomy. ApoA-IV mRNA expression was analyzed by real-time polymerase chain reaction (PCR) to exclude de novo synthesis in the kidney. RESULTS: ApoA-IV immunostaining was detected in proximal and distal tubular cells, capillaries and blood vessels but not inside glomeruli. ApoA-IV was predominantly found in the brush border of proximal tubules and in intracellular granules and various plasma membrane domains of both proximal and distal tubules. mRNA expression analysis revealed that no ApoA-IV was produced in the kidney. CONCLUSION: The immunoreactivity of ApoA-IV observed in kidney tubular cells suggests a direct role of the human kidney in ApoA-IV metabolism. The granular staining pattern probably represents lysosomes degrading ApoA-IV. The additional ApoA-IV localization in distal tubules suggests a rescue function to reabsorb otherwise escaping ApoA-IV in case proximal tubules cannot reabsorb all ApoA-IV. Since no mRNA expression could be detected in any kidney cells, the observed ApoA-IV immunoreactivity represents uptake and not de novo synthesis of ApoA-IV.

Immune-regulation of the apolipoprotein A-I/C-III/A-IV gene cluster in experimental inflammation.

Apolipoprotein A-IV is a member of the apo A-I/C-III/A-IV gene cluster. In order to investigate its hypothetical coordinated regulation, an acute phase was induced in pigs by turpentine oil injection. The hepatic expression of the gene cluster as well as the plasma levels of apolipoproteins were monitored at different time periods. Furthermore, the involvement of the inflammatory mediators' interleukins 1 and 6 and tumor necrosis factor in the regulation of this gene cluster was tested in cultured pig hepatocytes, incubated with those mediators and apo A-I/C-III/A-IV gene cluster expression at the mRNA level was measured. In response to turpentine oil-induced inflammation, a decreased hepatic apo A-IV mRNA expression was observed (independent of apo A-I and apo C-III mRNA) not correlating with the plasma protein levels. The distribution of plasma apo A-IV experienced a shift from HDL to larger particles. In contrast, the changes in apo A-I and apo C-III mRNA were reflected in their corresponding plasma levels. Addition of cytokines to cultured pig hepatocytes also decreased apo A-IV and apo A-I mRNA levels. All these results show that the down-regulation of apolipoprotein A-I and A-IV messages in the liver may be mediated by interleukin 6 and TNF-alpha. The well-known HDL decrease found in many different acute-phase responses also appears in the pig due to the decreased expression of apolipoprotein A-I and the enlargement of the apolipoprotein A-IV-containing HDL.

Apolipoproteins A-IV and A-V are acute-phase proteins in mouse HDL.

BACKGROUND: Infection and inflammation are associated with atherosclerosis. During infection and inflammation, HDL decreases and there are changes in the levels of several HDL-associated proteins. To identify changes in the protein composition of HDL during infection and inflammation, a proteomic approach was utilized. METHODS AND RESULTS: Using two-dimensional gel electrophoresis and mass spectrometry, we found the expected increases in apolipoprotein (apo) SAA and apo E, as well as a decrease in apo A-I on HDL isolated from mice injected with endotoxin. We identified apo A-IV and apo A-V as positive acute-phase proteins in mouse HDL. We also found an increase in hepatic mRNA levels of apo A-IV and apo A-V after injection of endotoxin. Interleukin-6 increased apo A-IV and apo A-V mRNA levels in Hep3B cells. Additionally, we demonstrated that the protein levels of apo A-II in acute-phase HDL and the hepatic mRNA levels of apo A-II were decreased. CONCLUSIONS: Apo A-IV and A-V are positive acute-phase proteins that increase in the serum during inflammation while apo A-II is a negative acute-phase protein in mice. Similar to other positive and negative acute-phase proteins, changes in hepatic production account for the changes in serum levels. However, the changes in apo A-IV and apo A-V, two apolipoproteins whose activities are not fully understood, may serve functions other than regulating lipid metabolism during the acute-phase response (APR). Coupled with the other changes in HDL proteins that occur, these changes are likely to alter the functional properties of HDL perhaps increasing the risk of atherosclerosis.

Apolipoprotein A-IV inhibits experimental colitis.

The antiatherogenic properties of apoA-IV suggest that this protein may act as an anti-inflammatory agent. We examined this possibility in a mouse model of acute colitis. Mice consumed 3% dextran sulfate sodium (DSS) in their drinking water for 7 days, with or without daily intraperitoneal injections of recombinant human apoA-IV. apoA-IV significantly and specifically delayed the onset, and reduced the severity and extent of, DSS-induced inflammation, as assessed by clinical disease activity score, macroscopic appearance and histology of the colon, and tissue myeloperoxidase activity. Intravital fluorescence microscopy of colonic microvasculature revealed that apoA-IV significantly inhibited DSS-induced leukocyte and platelet adhesive interactions. Furthermore, apoA-IV dramatically reduced the upregulation of P-selectin on colonic endothelium during DSS-colitis. apoA-IV knockout mice exhibited a significantly greater inflammatory response to DSS than did their WT littermates; this greater susceptibility to DSS-induced inflammation was reversed upon exogenous administration of apoA-IV to knockout mice. These results provide the first direct support for the hypothesis that apoA-IV is an endogenous anti-inflammatory protein. This anti-inflammatory effect likely involves the inhibition of P-selectin-mediated leukocyte and platelet adhesive interactions.

Apolipoprotein (a) mediates the lipoprotein (a)-induced biphasic shift in human platelet cyclic AMP.

Lipoproteins are known to influence platelet cyclic adenosine monophosphate (c-AMP) levels. Lipoprotein (a) (Lp(a))'s impact on platelet c-AMP levels has never been assessed. Increasing levels of purified human Lp(a) (1-100) mg/dl were incubated with washed human platelets. Lp(a) concentrations of 1-25 mg/dl resulted an initial statistically significant increase of platelet c-AMP above basal levels and decreased collagen-stimulated platelet aggregation levels. Higher concentrations progressively returned the platelet c-AMP concentrations to basal levels accompanied by further decreases in platelet aggregation. Increasing concentrations of purified apolipoprotein (a) (apo(a)) also resulted in a similar biphasic c-AMP response while Lp(a) without apo(a) was without impact. One antibody directed against apo(a) in intact Lp(a) removed the biphasic c-AMP pattern and eliminated Lp(a) platelet aggregation. Antibodies directed against apo B in intact Lp(a) gave results similar to intact Lp(a) in terms of the biphasic response of c-AMP upon platelet exposure to increasing levels of Lp(a). It is concluded that apo(a) mediates the Lp(a)-induced biphasic response in platelet c-AMP as the result of platelet exposure to increasing levels of Lp(a). The biphasic response in c-AMP assists in platelet aggregation decreases up to a concentration of 25 mg/dl Lp(a), such assistance being lost at higher Lp(a) concentrations.

Lp(a) particles mold fibrin-binding properties of apo(a) in size-dependent manner: a study with different-length recombinant apo(a), native Lp(a), and monoclonal antibody.

OBJECTIVE: Small-sized apolipoprotein(a) [apo(a)] isoforms with high antifibrinolytic activity are frequently found in cardiovascular diseases, suggesting a role for apo(a) size in atherothrombosis. To test this hypothesis, we sought to characterize the lysine (fibrin)-binding function of isolated apo(a) of variable sizes. METHODS AND RESULTS: Recombinant apo(a) [r-apo(a)] preparations consisting of 10 to 34 kringles and a monoclonal antibody that neutralizes the lysine-binding function were produced and used in parallel with lipoprotein(a) [Lp(a)] particles isolated from plasma in fibrin-binding studies. All r-apo(a) preparations displayed similar affinity and specificity for lysine residues on fibrin regardless of size (K(d) 3.6+/-0.3 nmol/L) and inhibited the binding of plasminogen with a similar intensity (IC50 16.8+/-5.4 nmol/L). In contrast, native Lp(a) particles displayed fibrin affinities that were in inverse relationship with the apo(a) kringle number. Thus, a 15-kringle apo(a) separated from Lp(a) and a 34-kringle r-apo(a) displayed an affinity for fibrin that was higher than that in the corresponding particles (K(d) 2.5 versus 10.5 nmol/L and K(d) 3.8 versus 541 nmol/L, respectively). However, fibrin-binding specificity of the r-apo(a) preparations and the Lp(a) particles was efficiently neutralized (IC50 0.07 and 4 nmol/L) by a monoclonal antibody directed against the lysine-binding function of kringle IV-10. CONCLUSIONS: Our data indicate that fibrin binding is an intrinsic property of apo(a) modulated by the composite structure of the Lp(a) particle.

Kringles of the plasminogen--prothrombin gene family share conformational epitopes with recombinant apolipoprotein (a): specificity of the fibrin-binding site.

Monoclonal antibodies directed against recombinant apolipoprotein (a) (r-apo(a)) lacking plasminogen-like KIV-2 repeats were used to identify structurally related conformational epitopes in various members of the plasminogen-prothrombin gene family. A number of procedures including a fibrin-binding inhibition immunoassay and surface plasmon resonance studies were used. Two antibodies (A10.1 and A10.4) recognised common conformational structures in r-apo(a), prothrombin, factor XII, plasminogen and its tissue-type and urokinase-type activators. In contrast, two other antibodies recognised specifically an epitope comprising residues of the lysine-binding site (A10.2) or close to it (A10.5) and inhibited the fibrin-binding function of r-apo(a) (IC(50)=36 pmol/l and 9.76 nmol/l, respectively). Interestingly, these antibodies distinctly recognised the elastase-derived fragments of plasminogen K4 (A10.2) and K1+2+3 (A10.5) without affecting plasminogen binding to fibrin. These results suggest that highly conserved conformational regions are common to various proteins of the plasminogen-prothrombin gene family and are in agreement with the concept that these proteins constitute a monophyletic group derived from an ancestral gene.

Production and characterization of monoclonal antibodies directed to the kringle V and protease domains of human apolipoprotein(a).

Production and use of anti-apolipoprotein(a) monoclonal antibodies (MAbs) specific to single copy regions in the polymorphic lipoprotein(a) (Lp(a)) has been emphasized to be important for the standardization of measurements of the coronary heart disease risk factor, Lp(a). Here, mouse MAbs were prepared against the kringle V (V) and protease (P) domains of human apolipoprotein(a) (apo(a)), which domains are present in single copy in the apo(a) molecule. The cDNA for apo(a)VP was cloned from human liver cDNA library, and the V-P recombinant protein overexpressed in Escherichia coli was used as an antigen for the antibody production. Two antibodies named as MAb(a)20 and MAb(a)23 were finally produced, and they were characterized for their binding specificity and epitopes. The specificity of the antibodies was confirmed by an immunoblotting procedure and an enzyme-linked immunoassay (ELISA). It was shown that the antibodies had little, if any, cross-reactivity with human plasminogen, which is relatively abundant in human serum and is highly homologous (85%) with apo(a) in amino acid (aa) sequence. For epitope analysis, 3'-deletional series of apo(a)VP cDNA were constructed, and expression products of them were analyzed for the binding MAb(a)20 and MAb(a)23 do. It has been revealed that distinct epitopes were recognized by the two MAbs: MAb(a)23 (gamma2b, kappa) bound to the V region about 60 aa downstream from the N-terminal, and MAb(a)20 (gamma1, kappa) bound to the P region close to the C-terminal. A one step-sandwich ELISA system for Lp(a) was developed using MAb(a)20 as a capturing antibody and horseradish peroxidase (HRP)-coupled MAb(a)23 as a detecting antibody. The assay was found to be sensitive and useful for detecting Lp(a) in the range of 4-150 microg/dL (80 pM-3 nM).

[A novel Lp(a) assay not affected by apo(a) size polymorphism].

Apo(a), the protein moiety of Lp(a), has size polymorphism that derives from the variable number of K4 type 2 repeats. We have developed three ELISA method with using monoclonal antibodies against the K4 type 2 repeats, the K4 type 5-protease domain, and the K5 protease domain of the apo(a) molecule. The commercial assay kits were correlated with the results from ELISA that utilized a monoclonal antibody against the K4 type 2 but indicated poor correlation with an ELISA method using anti Lp(a) K4 type 5-protease domain monoclonal antibody. Serum Lp(a) values were 1.8-fold overestimated by the commercial assay kits in samples with S4 type of the largest isoform, but our ELISA method in which the apo(a) K4 type 5-protease domain is used as labeled antibody and capture antibody was not affected by size polymorphism.

Producción de los reactivos primarios de un sistema inmunoenzimático ELISA para determinar lipoproteína (a) / Production of primary reactives of an ELISA immunoenzymatic system to determine lipoprotein (a)

El objetivo de nuestro trabajo fue desarrollar la metodología y la producción de los reactivos primarios para realizar un método inmunoenzimático (ELISA) tipo sandwich para determinar lipoproteina (a) en suero humano. La concentración final del antisuero policlonal antiapolipoproteína (a) (anti-apo [a]) obtenida fue de 1,3 mg/dL, purificado, por cromatografía de afinidad y de intercambio iónico. El antisuero antilipoproteína de baja densidad (anti LDL) de carnero purificado por cromatografía de afinidad fue utilizado en la obtención del conjugado policlonal perxodasa-IgG anti-LDL. La concentración óptima de recubrimiento con anti-apo (a) fue de 2 µg/mL y la dilución óptima del conjugado fue 1/7000, con lo cual obtuvimos una sensibilidad alta del ensayo. Poder producir en nuestro laboratorio reactivos primarios para el desarrollo de un sistema inmunoenzimático de determinación de Lp (a) hace posible la accesibilidad de la determinación con fines asistenciales e investigativos, así como un ahorro en moneda libremente convertible, pues estos reactivos tienen un elevado costo en el mercado(AU)

Producción de los reactivos primarios de un sistema inmunoenzimático ELISA para determinar lipoproteína (a) / Production of primary reactives of an ELISA immunoenzymatic system to determine lipoprotein (a)

El objetivo de nuestro trabajo fue desarrollar la metodología y la producción de los reactivos primarios para realizar un método inmunoenzimático (ELISA) tipo sandwich para determinar lipoproteina (a) en suero humano. La concentración final del antisuero policlonal antiapolipoproteína (a) (anti-apo [a]) obtenida fue de 1,3 mg/dL, purificado, por cromatografía de afinidad y de intercambio iónico. El antisuero antilipoproteína de baja densidad (anti LDL) de carnero purificado por cromatografía de afinidad fue utilizado en la obtención del conjugado policlonal perxodasa-IgG anti-LDL. La concentración óptima de recubrimiento con anti-apo (a) fue de 2 µg/mL y la dilución óptima del conjugado fue 1/7000, con lo cual obtuvimos una sensibilidad alta del ensayo. Poder producir en nuestro laboratorio reactivos primarios para el desarrollo de un sistema inmunoenzimático de determinación de Lp (a) hace posible la accesibilidad de la determinación con fines asistenciales e investigativos, así como un ahorro en moneda libremente convertible, pues estos reactivos tienen un elevado costo en el mercado

Factors influencing Lp[a]- particle size as determined by gradient gel electrophoresis.

This study examined factors influencing the particle diameter of Lp[a]-, the low density lipoprotein (LDL)-like moiety of Lp[a], in 26 subjects chosen to provide a range of Lp[a] and triglyceride levels. Lp[a] and LDL fractions were isolated by vertical density ultracentrifugation. Lp[a] was further purified using a lysine-Sepharose affinity column and Lp[a]- obtained by incubating Lp[a] with dithiothreitol. Lp[a], LDL, and Lp[a]- fractions were run on 3-13% gradient gels to determine particle diameter. Lp[a] size correlated positively with LDL size (r = 0.62; P < 0.001), but the association between Lp[a]- size and LDL size was stronger (r = 0.82; P < 0.0001). Log triglyceride level correlated inversely with Lp[a]- size (r = -0.72; P < 0.0001) and LDL size (r = 0.69; P < 0.0001). HDL cholesterol level correlated positively with Lp[a]- size (r = 0.67; P < 0.0005) and LDL size (r = 0.64; P < 0.0005). The strong correlation between LDL size and Lp[a]- size may be due to extracellular utilization of circulating LDL in the production of Lp[a] or may reflect the same metabolic processes influencing both these particles once Lp[a] has been formed.

A cytotoxic electronegative LDL subfraction is present in human plasma.

By using fast protein liquid chromatography, we isolated from human plasma a minor electronegative LDL subfraction designated LDL(-). After immunoaffinity chromatography against apolipoprotein (apo)(a) and apo A-I, LDL(-) represented 6.7 +/- 0.9% (mean +/- SD; n = 18) of total LDL. Compared with the major LDL subfraction, designated LDL(+), LDL(-) contained similar amounts of thiobarbituric acid-reactive substances, conjugated dienes, and vitamin E and had a similar lipid/protein ratio and mean density. Moreover, the apo B of LDL(-) was not aggregated and its LDL receptor-binding activity was slightly increased. These results were consistent with the nonoxidized nature of LDL(-). LDL(-) showed increased contents of sialic acid (38.1 +/- 5.2 versus 28.9 +/- 3.3 nmol/mg protein; n = 7; P < .01), apo C-III (1.43 +/- 0.21% versus 0.14 +/- 0.04%; n = 7; P < .01), and apo E (1.64 +/- 0.26% versus 0.10 +/- 0.05%; n = 7; P < .0005). Compared with LDL(+), LDL(-) displayed enhanced cytotoxic effects on cultured human umbilical vein endothelial cells, as shown by lactate dehydrogenase assay (P < .003; n = 6), neutral red uptake (P < .02; n = 6), and morphological studies. We also studied the relationship of LDL(-) to age and plasma lipid levels in 133 subjects. The percentage of contribution of LDL(-) to total plasma LDL correlated with age (P < .05), total cholesterol (P < .05), and LDL cholesterol (P < .003). In conclusion, this study shows that LDL(-), a circulating human plasma LDL, is an electronegative native LDL subfraction with cytotoxic effects on endothelial cells. This subfraction, which correlates positively with common atherosclerotic risk factors, might induce atherogenesis by actively contributing to alteration of the vascular endothelium.