Biomedical subjects

W Jessup

Publications and source records attributed to W Jessup.

At least 55 records · Page 3Linked to original sources

PubMed1997

Regulation of serum-induced lipid accumulation in human monocyte-derived macrophages by interferon-gamma. Correlations with apolipoprotein E production, lipoprotein lipase activity and LDL receptor-related protein expression.

The demonstration of lipid loaded macrophages in atherosclerotic tissue has led to the development of in vitro systems to elucidate the mechanisms involved in lipid accumulation. Here we have characterised the changes which occur in human monocyte-derived macrophage (MDM) lipids during culture in either human serum (HS) or foetal calf serum (FCS). MDM cultured in HS were rapidly converted to lipid filled foam cells, as assessed using HPLC analysis and oil red-O staining and compared with the same cells grown in FCS. However, the lipids which accumulated were predominantly triglycerides with smaller amounts of unesterified cholesterol (UC) and only traces of cholesteryl esters (CE). alpha-Tocopherol (alpha-TocH) was present at higher levels in MDM cultured in HS compared to the same cells grown in FCS. MDM lipid accumulation was dependent on the triglyceride-rich lipoprotein (TGRL) fraction of human serum; accordingly, supplementation of FCS with human TGRL also induced MDM lipid accumulation. The relationships between cellular lipid accumulation and secretion of apolipoprotein E (apo E) and lipoprotein lipase (LPL) as well as expression of the low density lipoprotein receptor-related protein (LRP) were also examined. MDM lipid accumulation was associated with increased apo E secretion but did not alter extracellular LPL activity. The lipid accumulation which was induced by HS was potently inhibited (but not reserved) by the inflammatory cytokine interferon-gamma (IFN gamma), and this was associated with decreased apo E production, LPL secretion and expression of LRP. These studies reveal striking differences in the lipid composition of MDM cultured in either HS or FCS, and indicate that oil red-O staining is not necessarily associated with cholesteryl ester accumulation in human macrophages. Furthermore, the effect that serum-induced lipid accumulation has on the specific MDM functions studied should be appreciated when developing in vitro macrophage models.

Apolipoproteins E↗

PubMed1997

Effects of CSF-1 on cholesterol accumulation and efflux by macrophages.

To assess whether human monocyte-specific colony-stimulating factor (CSF-1) might influence atherogenesis, CSF-1-induced macrophage responses that might contribute to enhanced clearance of low-density lipoprotein (LDL) or modified LDL were investigated. Careful account was made of cell preservation and increases in cell volume and protein (representing increased cell surface area, and thus endocytically active membrane) during culture with CSF-1. This permitted distinction between selective and nonspecific effects of CSF-1, the latter paralleling increases in cellular mass and volume. CSF-1 enhanced mouse peritoneal macrophage survival in vitro during exposure to lipoprotein-deficient serum with or without native LDL or acetylated LDL (Ac-LDL), as judged by maintenance of cellular DNA and cell numbers. In the presence of copper-oxidized LDL (Ox-LDL), such effects were very slight. In all conditions, CSF-1 increased cellular protein content. CSF-1 increased the uptake of both Ac-LDL and Ox-LDL calculated per culture, but this was entirely explicable by the increased cell protein, indicating that there was no selective enhancement of scavenger receptor or other routes for uptake of the modified LDLs. Similarly, CSF-1 also increased the accumulation of cholesterol and its esters nonspecifically. CSF-1 did have a marked and specific effect on the composition of cholesterol esters, decreasing the proportion of polyunsaturated esters relative to monounsaturated and saturated esters. Finally, cholesterol efflux induced by apolipoprotein A1 from Ac-LDL-loaded macrophages was not influenced by CSF-1. Thus, the enhanced macrophage catabolism of modified LDLs by CSF-1 is part of a nonspecific action on the cells but could contribute to a reduction in circulating cholesterol, observed in some situations of CSF-1 presentation in humans.

Animals↗

PubMed1997

7-Hydroperoxycholesterol and its products in oxidized low density lipoprotein and human atherosclerotic plaque.

7-Hydroperoxycholesterols (7OOHs) are intermediates in cholesterol oxidation and potential cytotoxins. A normal-phase HPLC method with UV (205 nm) detection was developed that could resolve 7 alpha OOH, 7 beta OOH, 7-ketocholesterol (7K), and the epimeric 7-hydroxycholesterols (7OHs). 7OOH formation was investigated when LDL was exposed to four different oxidizing systems: Cu2+; Ham's F-10; mouse peritoneal macrophages in Ham's F-10; and a metal-independent peroxyl-radical generating system (AAPH). With all four oxidizing systems, 7OOH (both free and esterified, mostly as the beta-isomer) was the major oxysterol formed at early times, with 7K dominating at later stages (> or = 24 h) in Cu-oxLDL. When LDL was oxidized in the presence of cells there was transfer of free oxysterols from LDL to the cells. Negligible 7OOH, but significant amounts of 7OH, accumulated in the cells suggesting efficient cellular reduction of 7OOH. Lipid extracts from eight plaque samples obtained from patients undergoing carotid endarterectomy were analyzed. Only trace amounts of 7OOH (< 0.02% of total cholesterol) could be detected using this normal-phase HPLC method with UV detection or with a more sensitive reverse-phase method utilizing chemiluminescence detection. 7K was the major 7-oxygenated sterol detected, at least 20-fold in excess of that calculated for 7OOH, followed by 7 beta OH and 7 alpha OH. The trace concentrations of 7OOH in plaque indicate its lability in biological/cellular systems and may signify the ability of cells in the artery wall to metabolize it further.

Animals↗

PubMed1996

Hydroxypropyl-beta-cyclodextrin-mediated efflux of 7-ketocholesterol from macrophage foam cells.

Atherosclerosis involves the arterial accumulation of lipid-laden "foam cells" containing oxidized and unoxidized sterols and their esters (Mattsson-Hulten, L., Lindmark, H., Diczfalusy, U., Bjorkhem, I., Ottosson, M., Liu, Y., Bondjers, G., and Wiklund, O. (1996) J. Clin. Invest. 97, 461-8). Oxidized sterols are probably critical to atherogenesis because they inhibit cholesterol removal from cells and are cytotoxic. We recently reported that there is deficient induction of cellular cholesterol efflux by apolipoprotein A-I, the main initial acceptor of cellular cholesterol from macrophages loaded in vitro with oxidized low density lipoprotein (Kritharides, L., Jessup, W., Mander, E., and Dean, R. T. (1995) Arterioscler. Thromb. 15, 276-289). There was an even more marked impairment of the release of 7-ketocholesterol which is a major oxysterol in these cells and in human atherosclerotic lesions. Here we show that hydroxypropyl-beta-cyclodextrin can induce selective efflux of 7-ketocholesterol. Efflux of 7-ketocholesterol was time and concentration dependent, and the rate of its removal was 50-fold greater for hydroxypropyl-beta-cyclodextrin than for apolipoprotein A-I. Over a defined range of concentrations (0-5 mg/ml), efflux of 7-ketocholesterol was preferred over that of cholesterol and occurred without cell toxicity. Efflux of free 7-ketocholesterol was associated with decreased intracellular free and esterified 7-ketocholesterol. Hydroxypropyl-beta-cyclodextrin also enhanced efflux of other oxysterols. The physical solubilization of 7-ketocholesterol by the cyclodextrin was much greater than that of cholesterol, in accordance with its differential effects on efflux. These data highlight the importance of extracellular sterol solubilization in the efflux of cellular oxysterols and the mobilization of intracellular free and esterified oxysterol pools in macrophages loaded with oxidized low density lipoprotein. Synthetic sterol-solubilizing agents such as hydroxypropyl-beta-cyclodextrin are thus potential prototypes for the further development of oxysterol-removing agents.

2-Hydroxypropyl-beta-cyclodextrin↗

PubMed1996

Sterol efflux is impaired from macrophage foam cells selectively enriched with 7-ketocholesterol.

The aim of the present study was to investigate whether impairment of cholesterol efflux previously found from mouse peritoneal macrophages loaded with oxidized low density lipoprotein (OxLDL) could be ascribed to the presence of oxysterols in these cells. 7-Ketocholesterol (7KC), the major oxysterol present in OxLDL-loaded cells, was selectively incorporated into unoxidized LDL, which was subsequently acetylated to produce a high uptake form. Mouse macrophages incubated with 7KC-enriched acetylated LDL (7kAcLDL) did not reveal cytotoxicity judged by cell protein and trypan blue exclusion. A large proportion of cellular 7KC was esterified, indicating that it is a substrate for acyl CoA:cholesterol acyltransferase. Cholesterol efflux from mouse macrophages loaded with 7kAcLDL, using apoA-I as a sterol acceptor, was impaired in cells containing >50 nmol of 7KC/mg of cell protein compared with cells loaded with oxysterol-free acetylated LDL. Thus impairment of cholesterol efflux could be reproduced in cells loaded with 7kAcLDL containing similar proportions of 7KC as OxLDL. 7KC itself was exported very poorly, even when the levels of 7KC in the cells were low. These results suggest that oxysterols present in foam cells in vitro can affect reverse sterol transport and may be potentially important in foam cell formation in vivo.

Animals↗

PubMed1996

Oxidized lipoproteins and nitric oxide.

Nitric oxide has an important biological role as endothelium-derived relaxing factor, a key agent in the maintenance of normal vascular tone. It can also suppress lipoprotein oxidation, a potential anti-atherogenic property. However, in arteries subject to hypercholesterolemia or atherosclerosis, whereas nitric oxide synthesis is normal its biological activity is attenuated. This may be caused by its inactivation in the intima by components of oxidized lipoproteins acting both directly (by reaction with nitric oxide) and indirectly (by simulation of release of nitric oxide scavengers). Thus in hypercholesterolemia the normal balance between nitric oxide availability and lipoprotein oxidation is shifted to favour a self-reinforcing cycle of nitric oxide depletion and accelerated lipoprotein oxidation that may ultimately lead to atherosclerosis.

Arteriosclerosis↗

PubMed1996

Free and esterified oxysterol: formation during copper-oxidation of low density lipoprotein and uptake by macrophages.

We have defined the lipid composition of copper-oxidized LDL (Cu-oxLDL) and a macrophage-foam cell model generated by the uptake of this modified lipoprotein. An HPLC method previously developed by our group for the measurement of lipid oxidation products of LDL was extended to permit the analysis of an array of 7-ketocholesteryl esters. Gas chromatography was used for the quantitation of oxysterols (free and esterified) in Cu-oxLDL and their subsequent uptake by macrophages. LDL (1.0 mg protein/ml) was oxidized using Cu(II) (20 microM) for up to 48 h at 37 degrees C. Resident mouse peritoneal macrophages were incubated with 24 h Cu-oxLDL (50 micrograms/ml) for 24 h. In 24 h Cu-oxLDL, cholesterol comprised approximately 50% of total sterols, 7-ketocholesterol comprised approximately 30% with five other oxysterols comprising the remainder (7 alpha- and 7 beta-hydroxycholesterol, cholesterol alpha- and beta-epoxides, and 6 beta-hydroxycholesterol). Macrophages that were incubated with 24 h Cu-oxLDL displayed a profile of oxysterols remarkably similar to that of 24 h Cu-oxLDL itself. The majority of cholesteryl esters and 7-ketocholesteryl esters in Cu-oxLDL and in Cu-oxLDL-loaded macrophages contained fatty acyl chains which are presumed oxidized. This work represents a comprehensive survey of free and esterified oxysterols in Cu-oxLDL and Cu-oxLDL-loaded macrophages and provides a basis for exploring how oxysterols are metabolized by macrophages and authentic human foam cells, and how, in turn, these oxysterols influence cellular metabolism.

Animals↗

PubMed1995

Biological fate of amino acid, peptide and protein hydroperoxides.

In the course of searching for a suitable marker for studying protein oxidation, we have successfully elucidated the structures of three valine hydroperoxides, i.e. beta-hydroperoxyvaline, (2S,3S)-gamma-hydroperoxyvaline and (2S,3R)-gamma-hydroperoxyvaline, which are novel products of protein oxidation. The corresponding valine hydroxides were obtained by sodium borohydride reduction [Fu, Hick, Sheil and Dean (1995) Free Rad. Biol. Med. 19, 281-292]. We hypothesized that valine hydroxides might be the major biological degradation products of valine hydroperoxides and, as such, could be useful markers for the study of protein oxidation in vivo. The aim of this study was to investigate the fate of valine hydroperoxide in selected biological systems by the use of chemiluminescence detection of hydroperoxides and HPLC analysis of O-phthaldialdehyde derivatives of amino acid residues. The degradation of hydroperoxides present on gamma-radiolysed solutions of valine, Pro-Val-Gly, or BSA occurred in the presence of: (1) transition metals (Fe2+, Fe3+, or Cu2+), (2) the detoxifying enzyme GSH peroxidase, (3) human plasma, and (4) J774 mouse monocyte macrophage cells. The major degradation product of valine hydroperoxide recovered in each case was found to be a valine hydroxide. These results suggest that valine hydroxide (derived from the hydroperoxide) may well be a useful in vivo marker for studying protein damage under oxidative stress.

Amino Acid Sequence↗

PubMed1995

Macrophages require both iron and copper to oxidize low-density lipoprotein in Hanks' balanced salt solution.

The oxidation of low-density lipoprotein (LDL) may be important in the pathogenesis of atherosclerosis. However, the interactions between cells and metals in promoting LDL oxidation are inadequately understood. A sensitive high-performance liquid chromatography analysis of cholesterol, cholesteryl esters, and their oxidation products was used to identify and accurately measure LDL oxidation achieved in thiol-free Hanks' balanced salt solution (HBSS) at pH 7.4. Mouse peritoneal macrophages inhibited LDL oxidation when incubated in HBSS containing either 10 microM iron or 1 microM copper, but were markedly prooxidant in the presence of both metals. The prooxidant effect of macrophages in the presence of both iron and copper did not require the provision of added disulfides or thiols. Both Fe2+ and macrophages were demonstrated to independently reduce Cu2+ to Cu1+ in HBSS, indicating that the direct reduction of copper by cells or iron may underlie the observed promotion of LDL oxidation by macrophages in this system. We conclude that macrophages can either promote or inhibit metal-mediated LDL oxidation and that externally supplied thiols are not essential to the promotion of LDL oxidation by cells. The presence of both iron and copper may be particularly important for macrophages to promote LDL oxidation in vivo.

Animals↗

PubMed1995

Oral L-arginine inhibits platelet aggregation but does not enhance endothelium-dependent dilation in healthy young men.

OBJECTIVES: Our aim was to assess the effect of oral L-arginine on endothelial or platelet physiology in humans. BACKGROUND: L-Arginine is the substrate for nitric oxide synthesis, and in cholesterol-fed rabbits, oral L-arginine improves endothelium-dependent dilation, inhibits platelet aggregation and reduces atheroma. In hypercholesterolemic humans, intravenous L-arginine immediately improves endothelium-dependent dilation; however, the vascular effects of oral L-arginine in healthy humans have not previously been investigated. METHODS: In a prospective, double-blind, randomized crossover trial, 12 healthy young men 27 to 37 years old took L-arginine (7 g three times daily) or placebo for 3 days each, separated by a washout period of 7 to 14 days. RESULTS: After L-arginine, plasma levels of arginine (mean +/- SEM 303 +/- 36 vs. 128 +/- 12 mumol/liter, p = 0.01) and urea (6.7 +/- 0.5 vs. 5.2 +/- 0.2 mmol/liter, p < 0.01) were higher than levels measured after placebo, and platelet aggregation in response to adenosine diphosphate was markedly impaired (37 +/- 12% vs. 81 +/- 3%, p = 0.02). The inhibition of platelet aggregation correlated with the plasma level of L-arginine (r = 0.74, p = 0.01), and it could be completely or partially reversed by ex vivo incubation with N-monomethyl-L-arginine, a specific nitric oxide synthase inhibitor. Platelet cyclic guanosine monophosphate levels were higher after oral L-arginine than at baseline (1.91 +/- 0.46 vs. 1.38 +/- 0.40 pmol/10(9) platelets, p = 0.04). No changes were seen in fasting lipid levels, heart rate, blood pressure, endothelium-dependent dilation of the brachial artery (measured in response to reactive hyperemia, using external vascular ultrasound) (6.1 +/- 0.7% vs. 6.5 +/- 0.7%, p = NS) or in plasma levels of nitrosylated proteins (a marker of in vivo nitric oxide production) (3.5 +/- 0.5 vs. 3.3 +/- 0.4 mumol/liter, p = NS) 1 to 1.5 h after the last dose of L-arginine. CONCLUSIONS: In these healthy young adult men, oral L-arginine inhibited platelet aggregation by way of the nitric oxide pathway. However, it had no effect on systemic hemodynamic variables, plasma nitrosylated protein levels or endothelium-dependent dilation. Therefore, at certain doses, oral L-arginine may result in a relatively platelet-specific increase in nitric oxide production.

Administration, Oral↗

PubMed1995

Apolipoprotein A-I-mediated efflux of sterols from oxidized LDL-loaded macrophages.

Although oxidized low-density lipoprotein (Ox-LDL) can accumulate in macrophages in vitro, generating cholesterol-loaded cells, little attention has been paid to the capacity of such macrophages loaded with OxLDL to export cholesterol and oxidized sterol moieties. In vitro lipid-loaded cells were generated by incubating primary cultures of mouse peritoneal macrophages with acetylated LDL (AcLDL) or OxLDL for 24 hours. The cellular content of native cholesterol, individual cholesteryl esters, and 7-ketocholesterol was determined by high-performance liquid chromatography. These cells were then incubated with medium containing apolipoprotein (apo) A-I and albumin or albumin alone for up to 24 hours; cholesterol and oxidized sterol efflux were measured both in terms of intracellular depletion and extracellular accumulation. Macrophages loaded with AcLDL accumulated cholesterol and large quantities of cholesteryl esters, whereas OxLDL-loaded cells accumulated cholesterol, a number of oxidized compounds (predominantly 7-ketocholesterol), and a relatively small quantity of cholesteryl esters. AcLDL-derived cells released approximately 50% of their total cholesterol (unesterified and esterified) to apo A-I-containing medium over 24 hours in the form of unesterified cholesterol, whereas OxLDL-derived cells released approximately 30% of their total cholesterol and 7% of their total content of 7-ketocholesterol over the same period. There was minimal efflux of any sterol in the absence of apo A-I. The proportions of cholesterol and 7-ketocholesterol released by either AcLDL- or OxLDL-loaded cells were not reduced by inhibiting cellular acyl-CoA:cholesterol acyl transferase using Sandoz 58-035, despite substantial alterations in the proportions of both free cholesterol and (in OxLDL-loaded cells) free 7-ketocholesterol in these cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Batch-to-batch variation of Chelex-100 confounds metal-catalysed oxidation. Leaching of inhibitory compounds from a batch of Chelex-100 and their removal by a pre-washing procedure.

Removal of adventitious redox-active metals from buffers by treatment with Chelex resin is a widely used procedure in free radical research. Use of a new batch of Chelex-100 resin in our laboratory coincided with a sudden inability to oxidise low-density lipoprotein with copper. We found that copper-mediated oxidation of ascorbate in water treated with the same batch of Chelex was inhibited when compared with untreated water and water treated with a different batch of the resin. Washing the Chelex removed the inhibitory effect suggesting that material was leaching from the resin. The washing procedure for Chelex-100 described is simple and can be scaled up. Oxidation of ascorbate with low concentrations of copper can be used to test the quality of batches of the resin.

Catalysis↗

PubMed1995

EDTA differentially and incompletely inhibits components of prolonged cell-mediated oxidation of low-density lipoprotein.

The extent to which cells can oxidize LDL may be underestimated because of the use of standard and arbitrary 24 hour in vitro incubations of cells with LDL. Such incubations have resulted in inconsistent results regarding the ability of cell-mediated LDL oxidation to generate relatively advanced oxidation products such as 7-ketocholesterol (7-KC). We studied prolonged oxidation of low density lipoprotein (LDL) by mouse peritoneal macrophages using HPLC measurement of cholesterol, cholesteryl esters and their oxidation products 7-KC and cholesteryl linoleate hydroperoxide (CL-OOH). Cell-mediated oxidation in Ham's F10 consistently followed the successive stages previously described during 24 hour-10 microM copper-mediated LDL oxidation, always generating 7-KC if allowed to proceed for sufficient time. The degree of inhibition of LDL oxidation achieved by metal chelators EDTA and DTPA at more advanced stages of cell-mediated LDL oxidation was not predictable from the published effects of such chelators upon early stages of metal-mediated and cell-mediated LDL oxidation. EDTA and DTPA only incompletely prevented the consumption of cholesteryl esters and the loss of performed CL-OOH when added after cell-mediated LDL oxidation was established, while effectively concurrently inhibiting the generation of 7-KC. These data indicate that progressive cell-mediated peroxidation of LDL cholesteryl esters and decomposition of CL-OOH may be less dependent upon a continuing supply of redox active metals than is the generation of 7-KC. In addition, they confirm the plausibility of prolonged cell-mediated oxidation of LDL as a source of oxysterols found in human atherosclerotic plaque, and imply that active redox cycling of metals is particularly important for their generation in vivo.

Animals↗

PubMed1994

Human macrophage-mediated oxidation of low-density lipoprotein is delayed and independent of superoxide production.

There is growing evidence that oxidatively modified low-density lipoprotein (LDL) accumulates in the atherosclerotic intima of arteries. Cells present in the intima (including the monocyte/macrophage) are capable of oxidizing LDL in vitro, but the mechanisms by which this occurs are unknown. Several reports have claimed a crucial role for superoxide as a cell-derived radical species capable of enhancing the rate of LDL oxidation. We have used a sensitive h.p.l.c. system with chemiluminescence detection to measure LDL cholesteryl ester hydroperoxides at early stages of LDL oxidation. During the initial stages of LDL oxidation, there is at least a 2 h delay before human monocyte-derived macrophages enhance this process. Stimulation of these cells to produce large fluxes of superoxide does not increase the rate of LDL oxidation or decrease the delay of its onset. Prior exposure of LDL to a high flux of superoxide does not increase its susceptibility to oxidation by human monocyte-derived macrophages. We also show that the thiobarbituric acid-reactive substances (TBARS) assay does not always correlate with more direct methods of assessing LDL oxidation and confirm recent reports that superoxide dismutase only partially inhibits cell-mediated LDL oxidation. We conclude that superoxide does not play a major role in human monocyte-derived macrophage-mediated LDL oxidation under the conditions that we describe.

Cholesterol Esters↗

PubMed1994

Apolipoprotein B of oxidized LDL accumulates in the lysosomes of macrophages.

We have studied the intracellular fate of the apolipoprotein B of copper-oxidized LDL in cultured J774 macrophages, using subcellular fractionation and immunofluorescence techniques. The oxidized apolipoprotein B, using cell fractionation, was located primarily in secondary lysosomes (identified using the lysosomal marker-enzyme aryl sulfatase). Light microscopy using antibodies to the mannose-6-phosphate receptor, the lysosomal membrane protein lgp 120, and oxidized LDL (biotinylated) confirmed that apo B of oxidized LDL did accumulate in secondary lysosomes rather than in endosomes. We conclude from these results that the oxidized apolipoprotein B of LDL reaches the secondary lysosomes, but is not efficiently degraded, leading to intracellular accumulation within this compartment. If this occurs in vivo it may influence the physiology of the macrophage and their subsequent roles in forming foam cells and the development of the fatty streaks of early atherosclerosis.

Acylation↗

PubMed1994

Transendothelial transport of modified low-density lipoproteins.

Human umbilical vein endothelial cell monolayers were grown as monolayers on porous filters and their transcellular transport and degradation of 125I-labelled native and modified forms of LDL, supplied to either the intimal or the luminal face, were measured. Intact native, acetylated and oxidized LDL were all transported in both directions across the cell monolayers by receptor-independent mechanisms, and all forms of LDL were transported at similar rates. However, the mass of intact LDL transported from the intimal to the luminal face of the monolayer was always fourfold more than that transported in the opposite direction under similar conditions. In addition to LDL transport, endothelial cell monolayers also degraded native and modified forms of LDL by predominantly receptor-dependent routes, in that these could be inhibited (> 70%) by the addition of a 20-fold excess of the same form of (but unlabelled) LDL. The measured amounts of lipoprotein degraded were the same whether supplied to the intimal or the luminal face. Incubation of endothelial cells with oxidized LDL led to intracellular accumulation of a pool of macromolecular apo B which was apparently resistant to lysosomal proteolysis.

Biological Transport↗

PubMed1994

Enhanced LDL oxidation by murine macrophage foam cells and their failure to secrete nitric oxide.

Foam cells were produced in vitro by incubation of mouse peritoneal macrophages with acetylated or copper-oxidized LDL. Nitric oxide synthesis was stimulated by exposure of the cells to IFN gamma and LPS. Nitric oxide production, detected by measurement of nitrite in the culture medium, was unchanged in Ac-LDL loaded cells as compared with non-loaded cells. However, Ox-LDL foam cells produced 68-99% less nitrite than non-loaded cells. Failure to detect nitric oxide synthase (NOS) products from macrophages previously loaded with Ox-LDL appeared to result from lack of NOS activity, as little active enzyme could be recovered from Ox-LDL loaded cells. However, addition of Ox-LDL to an active cell-free NOS preparation had no direct effect on enzymic activity. When native LDL was subsequently incubated with these various IFN gamma/LPS stimulated cells, cells pre-loaded with Ox-LDL promoted, on average, a 2-fold greater increase in oxidative modification of the LDL added than either non-loaded or Ac-LDL loaded cells. That is, there was an inverse correlation between NOS activity and the ability of the cells to promote LDL oxidation. Unstimulated Ox-LDL loaded foam cells also oxidized LDL better than unstimulated non-loaded or Ac-LDL loaded foam cells, and the extent of oxidative modification was generally greater than seen with the equivalent IFN gamma/LPS stimulated cells. This suggests that Ox-LDL loading also affects some additional factor(s) responsible for cell-mediated LDL oxidation.

Acetylation↗

PubMed1993

A method for defining the stages of low-density lipoprotein oxidation by the separation of cholesterol- and cholesteryl ester-oxidation products using HPLC.

A new high-performance liquid chromatographic system for the identification of some of the lipid oxidation products of low-density lipoprotein (LDL) oxidized by copper is described. Using a reversed-phase C-18 column and an isocratic solvent system of acetonitrile/isopropanol/water (44/54/2, v/v/v), a number of oxidized lipid moieties were resolved and detected simply by their 234-nm absorbance. The nature of several of these compounds was determined by chromatographic criteria, chemiluminescence, and mass spectrometry. The production of compounds within 4 h oxidation corresponded to the production of lipid hydroperoxides, the quantitatively most important of which is cholesteryl linoleate hydroperoxide, and to the rapid decrease in the cholesteryl ester content of LDL detected at 210 nm. More prolonged copper oxidation (up to 48 h) of LDL resulted in decreased quantities of lipid hydroperoxide moieties and increased amounts of a number of other, nonhydroperoxide, compounds. 7-Ketocholesterol and cholesterol linoleate hydroxide are two of the major products of prolonged oxidation. The detection of oxidation products correlates with the modification of LDL protein, permits a four-stage definition of metal-mediated LDL oxidation, and enables the calculation of a quantitative index of oxidation (lipoprotein oxidation index). This method will be generally applicable to cell- and copper-mediated oxidation, and will enable standardization of, and direct comparison between, different preparations of oxidized LDL.

Apolipoproteins B↗