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E Malle

Publications and source records attributed to E Malle.

At least 37 records · Page 2Linked to original sources
PubMed2000

Role of serum amyloid A during metabolism of acute-phase HDL by macrophages.

The serum amyloid A (SAA) family of proteins is encoded by multiple genes that display allelic variation and a high degree of homology in mammals. Triggered by inflammation after stimulation of hepatocytes by lymphokine-mediated processes, the concentrations of SAA may increase during the acute-phase reaction to levels 1000-fold greater than those found in the noninflammatory state. In addition to its role as an acute-phase reactant, SAA (104 amino acids, 12 kDa) is considered to be the precursor protein of secondary reactive amyloidosis, in which the N-terminal portion is incorporated into the bulk of amyloid fibrils. However, the association with lipoproteins of the high-density range and subsequent modulation of the metabolic properties of its physiological carrier appear to be the principal role of SAA. Because SAA may displace apolipoprotein A-I, the major protein component of native high density lipoprotein (HDL), during the acute-phase reaction, the present study was aimed at (1) investigating binding properties of native and acute-phase (SAA-enriched) HDL by J774 macrophages, (2) elucidating whether the presence of SAA on HDL particles affects selective uptake of HDL-associated cholesteryl esters, and (3) comparing cellular cholesterol efflux mediated by native and acute-phase HDL. Both the total and the specific binding at 4 degrees C of rabbit acute-phase HDL were approximately 2-fold higher than for native HDL. Nonlinear regression analysis revealed K(d) values of 7.0 x 10(-7) mol/L (native HDL) and 3.1 x 10(-7) mol/L (acute-phase HDL), respectively. The corresponding B(max) values were 203 ng of total lipoprotein per milligram of cell protein (native HDL) and 250 ng of total lipoprotein per milligram of cell protein (acute-phase HDL). At 37 degrees C, holoparticle turnover was slightly enhanced for acute-phase HDL, a fact reflected by 2-fold higher degradation rates. In contrast, the presence of SAA on HDL specifically increased (1. 7-fold) the selective uptake of HDL cholesteryl esters from acute-phase HDL by J774 macrophages, a widely used in vitro model to study foam cell formation and cholesterol efflux properties. Although ligand blotting experiments with solubilized J774 membrane proteins failed to identify the scavenger receptor-BI as a binding protein for both native and acute-phase HDL, 2 binding proteins with molecular masses of 100 and 72 kDa, the latter comigrating with CD55 (also termed decay-accelerating factor), were identified. During cholesterol efflux studies, it became apparent that the ability of acute-phase HDL with regard to cellular cholesterol removal was considerably lower than that for native HDL. This was reflected by a 1.7-fold increase in tau/2 values (22 versus 36 hours; native versus acute-phase HDL). Our observations of increased HDL cholesteryl ester uptake and reduced cellular cholesterol efflux (acute-phase versus native HDL) suggest that displacement of apolipoprotein A-I by SAA results in considerable altered metabolic properties of its main physiological carrier. These changes in the apolipoprotein moieties appear (at least in the in vitro system tested) to transform an originally antiatherogenic into a proatherogenic lipoprotein particle.

Acute-Phase Reaction↗
PubMed1999

Hypochlorite modification of high density lipoprotein: effects on cholesterol efflux from J774 macrophages.

The present study was aimed at investigating effects of hypochlorite (HOCl) modification of high density lipoproteins subclass 3 (HDL3) on their ability for cellular cholesterol removal from permanent J774 macrophages. Our findings indicate that HOCl (added as reagent or generated enzymatically by the myeloperoxidase/H2O2/Cl- system) damages apolipoprotein A-I, the major protein component of HDL3. Fatty acid analysis of native and HOCl-modified HDL3 revealed that unsaturated fatty acids in both major lipid subclasses (phospholipids and cholesteryl esters) are targets for HOCl attack. HOCl modification resulted in impaired HDL3-mediated cholesterol efflux from J774 cells, regardless of whether reagent or enzymatically generated HOCl was used to modify the lipoprotein. Decreased cholesterol efflux was also observed after HOCl modification of reconstituted HDL particles. Impairment of cholesterol efflux from macrophages was noticed at low and physiologically occurring HOCl concentrations.

Animals↗
PubMed1999

Effects of cytokines, butyrate and dexamethasone on serum amyloid A and apolipoprotein A-I synthesis in human HUH-7 hepatoma cells.

Serum amyloid A (SAA) and apolipoprotein A-I (apo A-I) are secreted by the liver. As concentrations of both apolipoproteins are inversely related under normal and acute-phase conditions, human HUH-7 hepatoma cells were stimulated with interleukin (IL)-1alpha (100 and 200 U), IL-6 (50 and 100 U), butyrate (2 mM) and dexamethasone (2 x 10(-7)M and 1 x 10(-6)M), alone or in combination. Changes in SAA and apo A-I synthesis were monitored after metabolic labelling of the cells with [35S]-methionine. Intracellular and secreted SAA and apo A-I were immunoprecipitated, separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and the radioactivity in the corresponding bands was counted. Intracellular apolipoprotein levels were increased by all stimuli, either alone or in combination, between 2.7- and 5.5-fold (SAA) and between 2.8- and 4.1-fold (apo A-I), respectively. In a similar manner, apolipoprotein levels secreted by HUH-7 cells were increased between 3.1- and 4.3-fold (SAA) and between 1.9- and 3. 3-fold (apo A-I). Co-administration of cytokines, butyrate and/or dexamethasone had no pronounced synergistic effect on intracellular biosynthesis and secretion of SAA and apo A-I. The results from the present study suggest that apo A-I must not necessarily be considered as a negative acute-phase reactant.

Acute-Phase Reaction↗
PubMed1999

Effects of hypochlorite-modified low-density and high-density lipoproteins on intracellular Ca2+ and plasma membrane Ca(2+)-ATPase activity of human platelets.

The presence of hypochlorite-modified lipoproteins in atherosclerotic lesions suggests that HOCl, a naturally occurring oxidant formed by the myeloperoxidase-catalyzed reaction of H2O2 and Cl-, is a candidate for generation of modified lipoproteins in vivo. We have previously demonstrated that Cu(2+)-oxidized LDL inhibits platelet plasma membrane Ca(2+)-ATPase (PMCA) in isolated membranes and causes an increase in cytosolic Ca2+ in resting whole platelets. However, Cu(2+)-oxidized LDL may not be identical in structure and function to the physiologically modified lipoprotein. Since platelet function may be affected by native and modified lipoproteins, the effect of HOCl-modified LDL and HDL3 on platelet PMCA and on the free intracellular Ca2+ concentration ([Ca2+]i) of whole platelets has been investigated. We demonstrate that in contrast to Cu(2+)-oxidized LDL, HOCl-modified LDL and HDL3 stimulate platelet PMCA activity in isolated membranes and that this effect results in a decrease of [Ca2+]i in vivo. Thus, HOCl-oxidation produces modified lipoproteins with the potential for altering platelet function and with properties different from those of the Cu(2+)-oxidized counterparts.

Blood Platelets↗
PubMed1998

High-density lipoprotein (HDL3)-associated alpha-tocopherol is taken up by HepG2 cells via the selective uptake pathway and resecreted with endogenously synthesized apo-lipoprotein B-rich lipoprotein particles.

alpha-Tocopherol (alphaTocH) is transported in association with lipoproteins in the aqueous milieu of the plasma. Although up to 50% of circulating alphaTocH is transported by high-density lipoproteins (HDLs), little is known about the mechanisms of uptake of HDL-associated alphaTocH. During the current study, human apolipoprotein (apo)E-free HDL subclass 3 (HDL3) labelled with [14C]alphaTocH was used to investigate uptake mechanisms of HDL3-associated alphaTocH by a permanent hepatoblastoma cell line (HepG2). HDL3-associated alphaTocH was taken up independently of HDL3 holoparticles in excess of apoA-I comparable with the non-endocytotic delivery of cholesteryl esters to cells termed the 'selective' cholesteryl ester uptake pathway. Experiments with unlabelled HDL3 demonstrated net mass transfer of alphaTocH to HepG2 cells. Time-dependent studies with [14C]alphaTocH-labelled HDL3 revealed tracer uptake in 80-fold excess of apoA-I and in 4-fold excess of cholesteryl linoleate. In addition to HLDs, low-density lipoprotein (LDL)-associated alphaTocH was also taken up in excess of holoparticles, although to a lesser extent. These findings were confirmed with unlabelled lipoprotein preparations, in which HDL3 displayed a 2- to 3-fold higher alphaTocH donor efficiency than LDLs (lipoproteins adjusted for equal amounts of alphaTocH). An important factor affecting particle-independent uptake of alphaTocH was the cellular cholesterol content (a 2-fold increase in cellular cholesterol levels resulted in a 2.3-fold decrease in uptake). Pulse-chase studies demonstrated that some of the HDL3-associated alphaTocH taken up independently of holoparticle uptake was resecreted along with a newly synthesized apoB-containing lipoprotein fraction.

Acrylamide↗
PubMed1998

Cytokine regulation of the acute-phase protein levels in multiple myeloma.

BACKGROUND: Interleukin (IL) 6 has an important role in the regulation of acute-phase proteins (APPs) during an acute-phase response. We studied IL-6 and other cytokines to determine if they regulate serum APP levels in the same way under the condition of the aberrant, long-lasting 'acute-phase response' that occurs in patients with chronic inflammation and cancer. METHODS: Serum levels of nine positive APPs [CRP, SAA, C1-INH, Bf, C5, C8, C9, alpha 1-acidic glycoprotein (AGP) and haptoglobin] and two negative APPs [transferrin and alpha 2-HS glycoprotein (AHSG)] were measured using immunochemical methods in 59 multiple myeloma patients and in 72 healthy control subjects. Serum IL-6 and tumour necrosis factor (TNF) alpha levels were determined by bioassays. RESULTS: IL-6 was negatively correlated with five out of nine (C1-INH, C8, C9, AGP and haptoglobin) positive APPs but positively correlated with C-reactive protein (CRP). When patients with high and low IL-6 serum concentration were compared, CRP levels were higher, AGP and haptoglobin levels were lower in the high- than in the low-L-6 group, whereas no significant difference between the two groups was found in levels of the other positive and negative APPs. TNF-alpha levels were negatively correlated with transferrin and AHSG levels. No difference in the levels of positive APPs was observed between patients with low and high TNF-alpha serum concentration. By contrast, levels of both transferrin and AHSG were significantly lower in the high- than in the low-TNF-alpha group. CONCLUSIONS: These findings indicate that, except for regulation of the negative APPs by TNF-alpha, the mechanism of APP regulation is different under the conditions of the short-term and the chronic, long-lasting 'acute-phase reaction'.

Acute-Phase Proteins↗
PubMed1998

Mapping of antigenic determinants of purified, lipid-free human serum amyloid A proteins.

Serum amyloid A (SAA) is the major apolipoprotein of high-density lipoproteins (HDL) present during the acute-phase reaction. To map specific epitopes on purified, lipid-free SAA, sequence-specific antibodies raised against synthetic peptides corresponding to amino acid residues 1-17, 14-30, 27-44, 40-63, 59-72, 68-84, 79-94 and 89-104 of human SAA1 were studied. Using the indirect sandwich dissociation-enhanced lanthanide fluorescence immunoassay, antibodies raised against epitopes comprising residues 1-17, 14-30, 40-63 and 79-94 failed to recognize the corresponding domains on isolated human SAA1/SAA2 or a mixture of both isoforms, indicating that these epitopes are masked, apparently because of specific folding and/or self-aggregation (dimerization). The accessible antigenic determinants of isolated SAA are epitopes comprising residues 31-39, 64-78 and 95-104. The present findings indicate that: (i) the same epitopes are exposed, irrespective whether SAA is HDL-associated or in its lipid-free form and that (ii) monomeric and dimeric SAA co-exist to a similar extent in the lipid-free form, irrespective of whether conditions are non-denaturating, denaturating, acidic or basic. From our studies it is proposed that isolated, purified SAA may serve as a reliable standard for quantification of HDL-associated SAA and for mimicking the interaction of acute-phase HDL particles with peripheral tissues in vitro.

Epitope Mapping↗
PubMed1998

Effect of hypochlorite (HOCl)-modified low density lipoproteins and high density lipoproteins on platelet function.

As hypochlorous acid (HOCl) might be a potential candidate for generation of modified (lipo)proteins in vivo , the present study was aimed at investigating the effects of HOCl-modified lipoproteins on platelet function in vitro. Lipoproteins modified with HOCl at concentrations that occur physiologically did not induce spontaneous platelet aggregation. However, low density lipoproteins (LDL; 100 to 500 microg protein/ml) increased platelet aggregation and fluorescence anisotropy in response to ADP (1 and 10 microM) and thrombin (0.1 and 0.5 U/ml) as well as malondialdehyde (MDA) formation as a function of increasing HOCl-concentrations (0.2 to 1.6 mM). HOCl-modified high density lipoprotein subfraction 3 (HDL3) had no effect on platelet function.

Journal Article↗
PubMed1997

Serum amyloid A (SAA) protein enhances formation of cyclooxygenase metabolites of activated human monocytes.

As serum amyloid A (SAA), an apolipoprotein associated with HDL during the acute-phase reaction may induce Ca2+ mobilization in human monocytes we raised the question whether SAA1 the predominant isoform of human acute-phase SAA is able to alter eicosanoid formation. In resting monocytes SAA1 was without effect on the secretion of cyclooxygenase metabolites while in calcium ionophore A23187- (0.5 and 2.5 microM) stimulated cells SAA1 led to a pronounced dose-dependent increase of TXA2, PGE2, and PGF2alpha. In addition a time-dependent increase of cyclooxygenase metabolites in between 1.5- and 3-fold in the presence of SAA1 was observed; apo A-I, the main HDL-apolipoprotein under non-acute-phase conditions, had no effect. Using sequence-specific anti-human SAA1 peptide (40-63) F(ab)2 fragments we could show that the proposed Ca2+-binding tetrapeptide Gly48-Pro49-Gly50-Gys51 of SAA1 is not responsible for enhanced biosynthesis of cyclooxygenase metabolites. Finally, we could demonstrate that human SAA1 is unable to bind Ca2+-ions, suggesting that SAA1 does not directly enhance eicosanoid biosynthesis via Ca2+ mobilization leading to enhanced phospholipase A2 activity.

Dinoprost↗
PubMed1997

Effects of reagent and enzymatically generated hypochlorite on physicochemical and metabolic properties of high density lipoproteins.

Myeloperoxidase (MPO), a protein secreted by activated phagocytes, may be a potential candidate for the generation of modified/oxidized lipoproteins in vivo via intermediate formation of HOCl, a powerful oxidant. During the present study, the effects of reagent NaOCl and OCl- generated by the MPO/H2O2/Cl- system on physicochemical and metabolic properties of high density lipoprotein (HDL) subclass 3 (HDL3) were investigated. Up to a molar oxidant:lipoprotein ratio of approximately 30:1, apolipoprotein A-I (apoA-I), the major HDL3 apolipoprotein component, represented the preferential target for OCl- attack (consuming 35-76% of the oxidant), thereby protecting HDL3 fatty acids (consuming between 17 and 30% of the oxidant) against OCl--mediated modification. At molar oxidant:HDL3 ratios >/= 60:1, we have observed pronounced consumption of HDL3 unsaturated fatty acids with concomitant formation of fatty acid chlorohydrins. Modification of HDL3 in the presence of the MPO/H2O2/Cl- system resulted in amino acid oxidation in a manner comparable with that found with reagent NaOCl only. Treatment of HDL3 with reagent NaOCl as well as modification by the MPO/H2O2/Cl- system resulted in significantly enhanced turnover rates of HDL3 by mouse peritoneal macrophages, an effect that was not a result of HDL3 aggregation as judged by dynamic and static light-scattering experiments. In comparison with native HDL3, the degradation by macrophages was enhanced by 4- and 15-fold when HDL3 was modified with reagent NaOCl or the MPO/H2O2/Cl- system. Finally, the ability of HDL3 to promote cellular cholesterol efflux from macrophages was significantly diminished after modification with reagent NaOCl. Collectively, these results demonstrate that the modification of HDL3 by hypochlorite (added as reagent or generated by the MPO/H2O2/Cl- system) transformed an antiatherogenic lipoprotein particle into a modified lipoprotein with characteristics similar to lipoproteins commonly thought to initiate foam cell formation in vivo.

Animals↗
PubMed1997

Selective uptake of high density lipoprotein-associated cholesterylesters by differentiated Ob1771 adipocytes is modulated by endogenous and exogenous lipoprotein lipase.

The present study aimed at investigating mechanisms of selective uptake of HDL3-associated cholesterylesters (HDL3-CEs) by differentiated Ob1771 adipocytes. Our findings indicate that Ob1771 cells are capable of pronounced selective uptake of HDL3-CEs in 2.6-fold excess of HDL3 holoparticle uptake. Selective uptake of HDL3-CEs into a releasable pool (presumably located in the cellular plasma membrane) was temperature insensitive while prominent internalization into a non-releasable and subsequent hydrolysis in a non-chloroquine sensitive compartment occurred at 37 degrees C. Release of membrane bound endogenous LPL by heparin resulted in decreased HDL3 holoparticle, total CE and selective CE uptake. Accordingly, the addition of exogenous LPL to the culture medium resulted in increased holoparticle, total CE and selective CE uptake.

Adipocytes↗
PubMed1997

Quantification of apolipoprotein D by an immunoassay with time-resolved fluorescence spectroscopy.

Apolipoprotein D (apoD), also known as gross cystic disease fluid protein-24 (GCDFP-24), is a minor protein moiety of high-density lipoproteins in human plasma. ApoD is expressed in a subset of breast carcinomas and has been proposed as a tumor marker and prognostic indicator for breast cancer progression. Here we describe a new sensitive time-resolved fluorimetric immunoassay for quantification of human apoD in biological specimens using affinity-purified polyclonal anti-human apoD rabbit antibodies and Eu3+ as a specific probe. Both purified apoD and normal human pool-serum served as reliable primary and secondary standards in the direct sandwich dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA). Plasma apoD concentrations measured by the DELFIA were 99.6 +/- 32 microg/ml. The detection limit of the DELFIA procedure was 0.5 ng/ml after sample dilution of 1/8000. The intra-assay coefficient of variation averaged 3.5%, whereas the inter-assay coefficient of variation averaged 6.9%. The concentration of apoD in breast cyst fluids ranged from 6.82 to 28.37 mg/ml. Based on the low detection limit and the high specificity of the DELFIA procedure, we have applied this technique for the measurement of apoD in breast cancer cell supernatants. In estrogen-receptor positive cells, i.e., T-47D and ZR-75-1 cells, 42.6 +/- 1.4 and 2.7 +/- 0.2 ng apoD/ml supernatant after 4 days in culture without induction of apoD synthesis were measured. A comparison of the direct sandwich DELFIA procedure with an electroimmunoassay commonly used to assay apoD revealed correlation coefficients of 0.986 (serum) and 0.975 (cyst fluids). The present findings indicate that the direct sandwich DELFIA is appropriate for apoD quantification in plasma and breast cyst fluids. Furthermore, the technique should permit studies on the induction of apoD synthesis in the low picomolar range in different carcinoma cells to gain insight into the expression of this atypical apolipoprotein.

Apolipoproteins↗
PubMed1997

Immunological evidence for hypochlorite-modified proteins in human kidney.

Oxygen radicals and oxidatively modified proteins seem to participate in degenerative vascular and inflammatory diseases. Factors that contribute to the development of atherosclerosis, eg, oxidation of low-density lipoproteins (LDLs), may also contribute to glomerulosclerosis. Although the nature of the in vivo oxidants remains unknown, recent findings indicated that the myeloperoxidase (MPO)-H2O2-halide system could play an important role in modification of (lipo)proteins in human tissues. MPO, the enzyme responsible for hypochlorite (HOCl/OCl-) formation, is present in human atherosclerotic lesions and in inflammatory conditions. In the present study, MPO was identified by Western blot analysis and immunohistochemical technique in diseased human kidney either with primarily sclerotic or inflammatory lesions. Furthermore, the presence of HOCl-modified proteins was demonstrated in diseased renal tissues using a specific monoclonal antibody (clone 2D10G9), raised against HOCl-modified LDL, that does not cross-react with native LDL or Cu(2+)-, 4-hydroxynonenal-, or malondialdehyde-modified LDL. The antibody recognized HOCl-modified proteins in glomerular and tubulointerstitial inflammatory and fibrotic lesions and pronounced immunostaining was demonstrated in mononuclear cells. LDL or human serum albumin oxidized by HOCl in vitro, but not native LDL or human serum albumin, effectively competed with epitopes in diseased kidney for antibody binding. Western blot analysis in diseased kidney protein samples revealed at least two major proteins recognized by the anti-HOCl-modified protein monoclonal antibody. Densitometric evaluation of immunoreactive bands obtained under these conditions demonstrated that expression of HOCl-modified proteins is tightly coupled to expression of immunoreactive MPO in the same tissue samples. From our studies it is proposed that oxidation of proteins by HOCl might be a leading event in glomerular and tubulointerstitial injury. By this mechanism, mononuclear cells, a permanent source for MPO, may play a key role in the development of nephrosclerosis, glomerulo-clerosis, and tubulointerstitial fibrosis, respectively.

Adult↗
PubMed1997

Implications of endogenous and exogenous lipoprotein lipase for the selective uptake of HDL3-associated cholesteryl esters by mouse peritoneal macrophages.

To investigate the implications of endogenous LPL on selective uptake of HDL3-associated cholesteryl esters (HDL3-CEs) by mouse peritoneal macrophages (MPMs), we have performed uptake experiments with MPMs obtained from control mice and transgenic knockout animals expressing LPL exclusively in muscle but not in macrophages. The capacity for HDL3 holoparticle, total HDL3-CE, and selective HDL3-CEs was independent of the expression of functional endogenous LPL (161 vs. 187, 1251 vs. 1300, and 1900 vs. 1113 ng HDL3/mg cell protein; control and LPL-deficient macrophages, respectively). Both control and LPL-deficient macrophages displayed, however, pronounced capacity for total HDL3-CE uptake in excess of HDL3 holoparticle uptake exceeding particle uptake by 7-fold. Despite the fact that endogenous LPL was without any effect on selective uptake, the addition of exogenous LPL led to a significant increase in cellular selective HDL3-CE uptake. Upon addition of purified LPL, HDL3 holoparticle (internalization and degradation), total HDL3-CE, and selective HDL3-CEs, was increased up to 2-fold. HDL3 holoparticle binding to control and LPL-deficient MPMs at 4 degrees C was enhanced 2.7- and 2.6-fold, respectively, in the presence of LPL. The present results suggest that endogenous LPL is without effect on selective uptake of HDL3-CEs. In contrast, the addition of exogenous LPL enhanced selective uptake of HDL3-CEs along with HDL3 holoparticle uptake apparently by the proposed bridging function of the enzyme.

Animals↗
PubMed1996

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.

Animals↗
PubMed1996

Quantification of lipoprotein lipase (LPL) by dissociation-enhanced lanthanide fluorescence immunoassay. Comparison of immunoreactivity of LPL mass and enzyme activity of LPL.

Lipoprotein lipase (LPL) hydrolyses triglycerides in chylomicrons and in very low density lipoproteins. In this study, a new sensitive enzyme immunoassay, the dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA), for the quantification of immunoreactive LPL mass in biological specimens was developed. In the indirect sandwich DELFIA assay polyclonal anti-human or anti-bovine LPL IgGs were used as capture antibodies, monoclonal antibody (mAb) 5D2 and Eu(3+)-labelled goat anti-mouse IgG were used as detection antibodies. In the direct sandwich DELFIA assay, mAb 5D2 was used as capture and Eu(3+)-labelled mAb 5D2 as detection antibodies. Both purified bovine and human LPL proteins served as standards in the indirect and the direct DELFIA assay. Standard curves were linear between 0.1 and 1000 ng LPL/ml, assuring the sensitivity of the DELFIAs within this range. Mean values for immunoreactive LPL mass in normal individuals were found to be 40.3 +/- 14.4 ng/ml preheparin plasma and 334.1 +/- 71 ng/ml postheparin plasma. In patients affected with type I hyperlipoproteinemia 82.4 +/- 29.3 ng/ml (postheparin plasma) were determined. Coefficients of inter- and intra-assay variation were 4.3% and 6.2% on average. The correlation coefficient between the indirect and the direct DELFIA technique was 0.9694. The correlation coefficient between immunoreactive LPL mass (estimated by DELFIA) and LPL activity (estimated by the LPL activity assay) was 0.9345. Our data are consistent with the concept that LPL is active as a dimer. Dissociation of the LPL dimer into monomers is tightly coupled to both loss of immunoreactivity and enzyme activity of LPL.

Animals↗
PubMed1996

Presence of hypochlorite-modified proteins in human atherosclerotic lesions.

Oxidation of LDL may contribute to atherogenesis, though the nature of the in vivo oxidant(s) remains obscure. Myeloperoxidase, the enzyme responsible for hypochlorous acid/hypochlorite (HOCl) production in vivo, is present in active form in human atherosclerotic lesions, and HOCl aggregates and transforms LDL into a high-uptake form for macrophages in vitro. Here we demonstrate HOCl-modified proteins in human lesions using an mAb raised against HOCl-modified LDL that recognizes HOCl-oxidized proteins but does not cross-react with Cu2+-, malondialdehyde-, or 4-hydroxynonenal-modified LDL. This antibody detected significantly more material in advanced atherosclerotic lesions than normal arteries, even though azide and methionine were included during sample work-up to inhibit myeloperoxidase and to scavenge HOCl. The epitope(s) recognized was predominantly cell associated and present in monocyte/macrophages, smooth muscle, and endothelial cells. The intima and cholesterol clefts stained more heavily than the center of the thickened vessels; adventitial staining was apparent in some cases. Immunostaining was also detected in a very early lesion from an accident victim, beside healthy areas that were unreactive. LDL oxidized by HOCl in vitro, but not native LDL, effectively competed with the epitopes in lesions for antibody binding. Density centrifugation of plaque homogenates and Western blot analysis showed that, in the apo B-containing lipoprotein fraction, the mAb recognized protein(s) of molecular mass greater than apo B, similar to those produced during oxidation of LDL with HOCl in vitro. Three major proteins were recognized by the anti-HOCl-modified protein antibody but not by an anti-apo B antibody in the apo B-free fraction. Together, these results demonstrate HOCl-oxidized proteins in human atherosclerotic lesions, implicating this oxidant in LDL modification in vivo.

Arteriosclerosis↗