Semi-synthetic thymoquinone analogs: new prototypes as potential antihyperlipidemics in irradiated rats.

AIM: Thymoquinone (TQ), has been reported to possess strong antihyperlipidemic properties. However, a variety of serious side effects has been reported for TQ. The present study aimed to evaluate the potential antihyperlipidemic activity of newly synthesized TQ analogs. METHODS & RESULTS: first, novel TQ derivatives were studied against radiation-induced dyslipidemia in male rats. Second, the most promising sulfur derivatives (4-7), were further tested to elucidate their possible mechanism(s) of actions. Results showed that they possess Hydroxymethyl Glutaryl-Co A reductase inhibitory activity, as well as stimulatory effects on the activities of each of plasma Lecithin-Cholesterol Acyltransferase and lipoprotein lipase enzymes. CONCLUSION: TQ derivatives (4-7), could be considered as promising agents in pathologies implicating impaired lipid metabolism, preclinical evaluation is warranted. [Formula: see text].

Agonistic Human Antibodies Binding to Lecithin-Cholesterol Acyltransferase Modulate High Density Lipoprotein Metabolism.

Drug discovery opportunities where loss-of-function alleles of a target gene link to a disease-relevant phenotype often require an agonism approach to up-regulate or re-establish the activity of the target gene. Antibody therapy is increasingly recognized as a favored drug modality due to multiple desirable pharmacological properties. However, agonistic antibodies that enhance the activities of the target enzymes are rarely developed because the discovery of agonistic antibodies remains elusive. Here we report an innovative scheme of discovery and characterization of human antibodies capable of binding to and agonizing a circulating enzyme lecithin cholesterol acyltransferase (LCAT). Utilizing a modified human LCAT protein with enhanced enzymatic activity as an immunogen, we generated fully human monoclonal antibodies using the XenoMouse(TM) platform. One of the resultant agonistic antibodies, 27C3, binds to and substantially enhances the activity of LCAT from humans and cynomolgus macaques. X-ray crystallographic analysis of the 2.45 Å LCAT-27C3 complex shows that 27C3 binding does not induce notable structural changes in LCAT. A single administration of 27C3 to cynomolgus monkeys led to a rapid increase of plasma LCAT enzymatic activity and a 35% increase of the high density lipoprotein cholesterol that was observed up to 32 days after 27C3 administration. Thus, this novel scheme of immunization in conjunction with high throughput screening may represent an effective strategy for discovering agonistic antibodies against other enzyme targets. 27C3 and other agonistic human anti-human LCAT monoclonal antibodies described herein hold potential for therapeutic development for the treatment of dyslipidemia and cardiovascular disease.

Effects of Pu-erh tea aqueous extract (PTAE) on blood lipid metabolism enzymes.

Disorders of blood lipid metabolism are the primary risk factors for many diseases. Recently, the effect of Pu-erh tea on blood lipid metabolism has received increasing attention. However, the mechanism underlying its ability to regulate blood lipid metabolism is unclear. We set out to study this through assessing the effects of Pu-erh tea aqueous extract (PTAE) on the central enzymes of blood lipid metabolism, including lipoprotein-associated phospholipase A2 (Lp-PLA2), lecithin: cholesterol acyltransferase (LCAT), 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) and pancreatic lipase (PL). We find that the Lp-PLA2, HMRG and PL activities are inhibited by PTAE in a dose-dependent manner and that the LCAT activity tends to increase with increasing PTAE concentrations. Lineweaver-Burk plot analyses reveal that PTAE acts as a competitive inhibitor for HMGR and PL and as a noncompetitive inhibitor for Lp-PLA2. Moreover, we determine that its active ingredients include catechins, gallic acid, caffeine, free amino acids, and soluble sugar. However, the effect of each ingredient and whether any of them have synergistic effects are still unknown. The results suggest that Pu-erh tea has a potent ability to regulate blood lipid metabolism and knowledge of the mechanisms provides insights into its potential therapeutic application as an alternative hypolipidemic drug.

Coincubation of PON1, APO A1, and LCAT increases the time HDL is able to prevent LDL oxidation.

The inhibition of low-density lipoprotein (LDL) oxidation by high-density lipoprotein (HDL) is a major antiatherogenic property of this lipoprotein. This activity is due, in part, to HDL associated proteins. However, whether these proteins interact in the antioxidant activity of HDL is unknown. LDL was incubated with apolipoprotein A1 (apo A1), lecithin:cholesterol acyltransferase (LCAT), and paraoxonase-1 (PON1) alone or in combination, in the presence or absence of HDL under oxidizing conditions. LDL lipid peroxide concentrations were determined. Apo A1, LCAT, and PON1 all inhibit LDL oxidation in the absence of HDL and enhance the ability of HDL to inhibit LDL oxidation. Their effect was additive rather than synergistic; the combination of these proteins significantly enhanced the length of time LDL was protected from oxidation. This seemed to be due to the ability of PON1 to prevent the oxidative inactivation of LCAT. Apo A1, LCAT, and PON1 can all contribute to the antioxidant activity of HDL in vitro. The combination of apo A1, LCAT, and PON1 prolongs the time that HDL can prevent LDL oxidation, due, at least in part, to the prevention LCAT inactivation.

Protective effect of lemongrass oil against dexamethasone induced hyperlipidemia in rats: possible role of decreased lecithin cholesterol acetyl transferase activity.

OBJECTIVE: To evaluate the anti-hyperlipidemic activity of lemongrass oil against in dexamethasone induced hyperlipidemia in rats. METHODS: Administration of dexamethasone was given at 10 mg/kg, sc. to the adult rats for 8 d induces hyperlipidemia characterized by marked increase in serum cholesterol and triglyceride levels along with increase in atherogenic index. RESULTS: Lemongrass oil (100 and 200 mg/kg, po.) treatment has showed significant inhibition against dexamethasone hyperlipidemia by maintaining the serum levels of cholesterol, triglycerides and atherogenic index near to the normal levels and the antihyperlipidemic effect of the lemongross oil was comparable with atorvastatin 10 mg/kg, po. The possible mechanism may be associated with decrease in lecithin cholesterol acetyl transferase (LCAT) activity. CONCLUSIONS: These results suggested that Lemon gross oil possess significant anti-hyperlipidemic activity.

Relevance of the amino acid conversions L144R (Zaragoza) and L159P (Zavalla) in the apolipoprotein A-I binding site for haptoglobin.

The high-density lipoprotein apolipoprotein A-I (ApoA-I) stimulates the enzyme lecithin-cholesterol acyltransferase (LCAT) in the reverse cholesterol transport pathway. Two ApoA-I variants, Zaragoza (L144R) and Zavalla (L159P), are associated with low levels of HDL-cholesterol but normal LCAT activity. Haptoglobin interacts with ApoA-I, impairing LCAT stimulation. Synthetic peptides matching the haptoglobin-binding site of native or variant ApoA-I (native, P2a; variants, Zav-pep and Zar-pep) bound haptoglobin with different activity: Zar-pep>P2a>Zav-pep. They also differently rescued LCAT in vitro activity in the presence of haptoglobin (P2a=Zar-pep>Zav-pep). Therefore, both amino acid conversions affect haptoglobin binding and LCAT regulation. We highlight the role of haptoglobin in LCAT regulation in subjects with ApoA-I variants.

Cholesteryl ester transfer protein promotes the formation of cholesterol-rich remnant like lipoprotein particles in human plasma.

BACKGROUND: Cholesteryl ester transfer protein (CETP) is suggested to be involved in the cholesterol level in remnant like lipoprotein particles (RLP), but there is no direct evidence that CETP increases cholesterol-rich RLP in plasma. METHODS: Human plasma was incubated with or without HDL containing [(3)H]-labeled cholesteryl ester ([(3)H]CE), recombinant CETP or CETP inhibitors at 37 degrees C in vitro. RESULTS: The RLP-cholesterol (RLP-C) level increased time-dependently and the amount of RLP-C increase (DeltaRLP-C) by the incubation was positively correlated with triglyceride (TG) level in plasma (r=0.597, P=0.0070). [(3)H]CE in HDL was transferred to RLP fraction under 37 degrees C incubation, and the amount of [(3)H]CE transferred to RLP correlated significantly with DeltaRLP-C in plasma (r=0.611, P=0.0156). Human recombinant CETP enhanced the RLP-C increase, while CETP inhibitor JTT-705 and anti-human CETP monoclonal antibody inhibited both the RLP-C increase and [(3)H]CE transfer to RLP. On the other hand, an inhibition of lecithin: cholesterol acyltransferase (LCAT) did not affect the RLP-C increase. In triglyceride-rich lipoproteins (TRL) fraction, JTT-705 inhibited [(3)H]CE transfer to RLP more strongly than that to non-RLP. CONCLUSIONS: CETP promotes the formation of cholesterol-rich RLP through the transfer of CE from HDL to TRL and CETP inhibitors are useful to reduce RLP-C.

[Depression of serum esterase and lipoprotein lipase activities in acute and longitudinal actions of ethanol].

Esterase (LCAT) and lipoprotein lipase (LPL) activities of blood serum, and a range of blood serum lipoproteins (LP) in acute and chronic ethanol intoxication were examined in healthy persons and patients with alcohol abuse. Ethanol inhibited LCAT and LPL activities, and increased apoA-containing LP in blood serum. These changes are considered as a basis for depression of a direct and reverse transportation of cholesterol in blood circulation under the action of ethanol.

Importance of acyl-coenzyme A:cholesterol acyltransferase 1/2 dual inhibition for anti-atherosclerotic potency of pactimibe.

Pactimibe sulfate, [7-(2,2-dimethylpropanamido)-4,6-dimethyl-1-octylindolin-5-yl]acetic acid hemisulfate, a novel Acyl-coenzyme A:cholesterol acyltransferase (ACAT) inhibitor, was investigated in vitro and in vivo to characterize its potential. Pactimibe exhibited dual inhibition for ACAT1 and ACAT2 (concentrations inhibiting 50% [IC50s] at micromolar levels) more potently than avasimibe. Kinetic analysis revealed pactimibe is a noncompetitive inhibitor of oleoyl-CoA (Ki value: 5.6 microM). Furthermore, pactimibe markedly inhibited cholesteryl ester formation (IC50: 6.7 microM) in human monocyte-derived macrophages, and inhibited copper-induced oxidation of low density lipoprotein more potently than probucol. Pactimibe exerted potent lipid-lowering and anti-atherosclerotic effects in atherogenic diet-fed hamsters. At doses of 3 and 10 mg/kg for 90 days, pactimibe decreased serum total cholesterol by 70% and 72%, and aortic fatty streak area by 79% and 95%, respectively. Despite similar cholesterol lowering, fatty streak area reduction was greater by 10 mg/kg. These results suggest that ACAT1/2 dual inhibitor pactimibe has anti-atherosclerotic potential beyond its plasma cholesterol-lowering activity.

ApoA-I/phosphatidylcholine discs remodels fast-migrating HDL into slow-migrating HDL as characterized by capillary isotachophoresis.

OBJECTIVE: Capillary isotachophoresis (cITP) is a technique for characterizing plasma lipoprotein subfractions according to their electrophoretic charges. We used this technique to examine the mechanism by which apoA-I/phosphatidylcholine (POPC) discs increase pre-beta HDL. METHODS AND RESULTS: The cITP analysis was performed using plasma prestained with a lipophilic dye on a Beckman P/ACE MDQ system. Plasma from a patient with lecithin:cholesterol acyltransferase (LCAT) deficiency who had increased apoE-containing HDL was used to characterize the charge distribution of apoA-I/POPC discs. cITP analysis of apoB- and E-depleted plasma of the patient in the presence of apoA-I/POPC discs indicated two major subfractions of apoA-I/POPC discs with mobilities of triglyceride-rich lipoproteins (fast and slow apoA-I). Incubation of whole plasma from a normolipidemic subject in the presence of apoA-I/POPC discs caused a reduction in cITP fast (f)- and intermediate (i)-migrating HDL, and fast and slow apoA-I, and an increase in slow (s)-migrating HDL. The changes in cITP lipoprotein subfractions were not affected by the inhibition of LCAT activity. ApoA-I/POPC discs increased the fractional esterification rate of cholesterol in apoB-depleted plasma. CONCLUSION: ApoA-I/POPC discs remodeled cITP fHDL and iHDL to sHDL independent of LCAT activity.

Lipid transfer proteins (LTP) and atherosclerosis.

This review deals with four lipid transfer proteins (LTP): three are involved in cholesteryl ester (CE) synthesis or transport, the fourth deals with plasma phospholipid (PL) transfer. Experimental models of atherosclerosis, clinical and epidemiological studies provided information as to the relationship of these LTP(s) to atherosclerosis, which is the main focus of this review. Thus, inhibition of acyl-CoA:cholesterol acyltransferase (ACAT) 1 and 2 decreases cholesterol absorption, plasma cholesterol and aortic cholesterol esterification in the aorta. The discovery that tamoxifen is a potent ACAT inhibitor explained the plasma cholesterol lowering of the drug. The use of ACAT inhibition in humans is under current investigation. As low cholesteryl ester transfer protein (CETP) activity is connected with high HDL-C, several CETP inhibitors were tried in rabbits, with variable results. A new CETP inhibitor, Torcetrapib, was tested in humans and there was a 50-100% increase in HDL-C. Lecithin cholesterol acyl-transferase (LCAT) influences oxidative stress, which can be lowered by transient LCAT gene transfer in LCAT-/- mice. Phospholipid transfer protein (PLTP) deficiency reduced apo B production in apo E-/- mice, as well as oxidative stress in four models of mouse atherosclerosis. In conclusion, the ability to increase HDL-C so markedly by inhibitors of CETP introduces us into a new era in prevention and treatment of coronary heart disease (CHD).

Assignment of the binding site for haptoglobin on apolipoprotein A-I.

Haptoglobin (Hpt) was previously found to bind the high density lipoprotein (HDL) apolipoprotein A-I (ApoA-I) and able to inhibit the ApoA-I-dependent activity of the enzyme lecithin:cholesterol acyltransferase (LCAT), which plays a major role in the reverse cholesterol transport. The ApoA-I structure was analyzed to detect the site bound by Hpt. ApoA-I was treated by cyanogen bromide or hydroxylamine; the resulting fragments, separated by electrophoresis or gel filtration, were tested by Western blotting or enzyme-linked immunosorbent assay for their ability to bind Hpt. The ApoA-I sequence from Glu113 to Asn184 harbored the binding site for Hpt. Biotinylated peptides were synthesized overlapping such a sequence, and their Hpt binding activity was determined by avidin-linked peroxidase. The highest activity was exhibited by the peptide P2a, containing the ApoA-I sequence from Leu141 to Ala164. Such a sequence contains an ApoA-I domain required for binding cells, promoting cholesterol efflux, and stimulating LCAT. The peptide P2a effectively prevented both binding of Hpt to HDL-coated plastic wells and Hpt-dependent inhibition of LCAT, measured by anti-Hpt antibodies and cholesterol esterification activity, respectively. The enzyme activity was not influenced, in the absence of Hpt, by P2a. Differently from ApoA-I or HDL, the peptide did not compete with hemoglobin for Hpt binding in enzyme-linked immunosorbent assay experiments. The results suggest that Hpt might mask the ApoA-I domain required for LCAT stimulation, thus impairing the HDL function. Synthetic peptides, able to displace Hpt from ApoA-I without altering its property of binding hemoglobin, might be used for treatment of diseases associated with defective LCAT function.

Increased plasma lipid-poor apolipoprotein A-I in patients with coronary artery disease.

BACKGROUND: Pre-beta 1-HDL participates in a cyclic process involved in the retrieval of cholesterol from peripheral tissues. Although pre-beta 1-HDL can be measured by two-dimensional electrophoresis or crossed immunoelectrophoresis, these methods are time-consuming and require technical expertise. In this study, we separated plasma lipid-poor apolipoprotein A-I (apo A-I) by high-performance size-exclusion chromatography. METHODS: We measured plasma lipid-poor apo A-I in 20 male patients with coronary artery disease [CAD; mean (SD) age, 64.0 (18) years] and 15 male controls [54.7 (17) years] and in 7 female CAD patients [70.3 (7.7) years] and 9 female controls [65.1 (4.7) years]. RESULTS: Lipid-poor apo A-I was most stable when stored at -80 degrees C in the presence of aprotinin (final concentration, 50 kIU/L). The lipid-poor apo A-I concentration decreased during incubation at 37 degrees C, and this was not prevented by the addition of 2 mmol/L of the lecithin:cholesterol acyltransferase (LCAT) inhibitor 5,5'-dithiobis(2-nitrobenzoic acid). Lipid-poor apo A-I was significantly higher in CAD patients than in controls [38.3 (7.9) mg/L for male CAD patients vs 29.3 (7.3) mg/L for male controls; 43.3 (11) mg/L for female CAD patients vs 27.1 (7.4) mg/L for female controls (P <0.01 for both)]. There were no significant differences in LCAT activity or cholesteryl ester transfer protein (CETP) concentration between patients and controls. Moreover, the plasma lipid-poor apo A-I concentration was not significantly correlated with LCAT or CETP activities. CONCLUSIONS: Although the production of lipid-poor apo A-I in plasma is not fully understood, our results indicate that lipid-poor apo A-I could be used as a marker for arteriosclerosis and demonstrate that it is not identical to the pre-beta1-HDL measured by other methods.

HMG-CoA reductase inhibition reverses LCAT and LDL receptor deficiencies and improves HDL in rats with chronic renal failure.

Dyslipidemia is a prominent feature of chronic renal failure (CRF) and a major risk factor for atherosclerosis and the progression of renal disease. CRF-induced dyslipidemia is marked by hypertriglyceridemia and a shift in plasma cholesterol from HDL to the ApoB-containing lipoproteins. Several studies have demonstrated a favorable response to administration of 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors (statins) in CRF. This study was intended to explore the effect of statin therapy on key enzymes and receptors involved in cholesterol metabolism. Accordingly, CRF (5/6 nephrectomized) and sham-operated rats were randomized to untreated and statin-treated (rosuvastatin 20 mg x kg(-1) x day(-1)) groups and observed for 6 wk. The untreated CRF rats exhibited increased total cholesterol-to-HDL cholesterol ratio, diminished plasma lecithin:cholesterol acyltransferase (LCAT) and the hepatic LDL receptor, elevated hepatic acyl-CoA:cholesterol acyltransferase (ACAT), and no change in hepatic HMG-CoA reductase, cholesterol 7alpha-hydroxylase, or HDL receptor (SRB-1). Statin administration lowered HMG-CoA reductase activity, normalized plasma LCAT, total cholesterol-to-HDL cholesterol ratio, and hepatic LDL receptor but did not significantly change either plasma total cholesterol, hepatic cholesterol 7alpha-hydroxylase, total ACAT activity, or SRB-1 in the CRF animals. Statin administration to the normal control rats led to significant increases in plasma LCAT and hepatic LDL receptor, significant reductions of total cholesterol-to-HDL cholesterol ratio, hepatic HMG-CoA reductase activity, and cholesterol 7alpha-hydroxylase abundance with virtually no change in plasma cholesterol concentration. Thus administration of rosuvastatin reversed LCAT and LDL receptor deficiencies and promoted a shift in plasma cholesterol from ApoB-containing lipoproteins to HDL in CRF rats.

Effect of simvastatin on trioleylglycerol hydrolysis and transacylation with cholesterol in serum of outpatients with coronary heart disease.

At present, the most effective drugs in treating hypercholesterolemia and atherosclerosis are the statins, which are potent inhibitors of the rate-limiting enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. Serum triacylglycerol (TAG) levels associate positively with the risk for coronary heart disease (CHD). Triacylglycerols are mainly hydrolyzed by the enzyme lipase (glycerol ester hydrolase [GEH], EC 3.1.1.3) but can also be transformed by transacylation with cholesterol (glycerol ester:cholesterol acyltransferase [GECAT], EC 2.3.1.43). We evaluated the effect of a 3-month treatment with simvastatin (10 mg/day) on GEH and GECAT activity in the serum of 26 outpatients with CHD. The activity of both GEH and GECAT was reduced in the CHD group compared with that in the control group: 5.9 +/- 0.9 mU/mg vs. 7.5 +/- 1.8 mU/mg and 11.1 +/- 1.4 mU/mg vs. 19.3 +/- 3.3 mU/mg, respectively (p < or = 0.05). In addition to the well known effect of reducing total cholesterol and low-density lipoprotein cholesterol in patients with CHD, we observed two other results of simvastatin treatment. First, GEH activity increased to values similar to those found in healthy subjects and, simultaneously, GECAT activity decreased. Trioleylglycerol transacylation with cholesterol amounted to 72% and hydrolysis to 28% in the control group and to 65% and 35% in the CHD group, respectively. After simvastatin treatment, transacylation with cholesterol and hydrolysis amounted to 51% and 49%, respectively. In conclusion, by increasing GEH and reducing GECAT, simvastatin seems not only to affect cholesterol synthesis but also to alter triacylglycerol metabolism. Further studies are needed to determine the physiological significance of these changes and their relationship with the development of atherosclerosis.

Endotoxin-lipoprotein complex formation as a factor in atherogenesis: associations with hyperlipidemia and with lecithin:cholesterol acyltransferase activity.

A potential role of endotoxin-lipoprotein (bacterial lipopolysaccharide-lipoprotein, LPS-LP) complex formation as a pathogenic factor for atherosclerosis has not been studied yet. The aim of this study was to test the hypothesis that in endotoxinemia in humans hyperlipidemia associated with atherosclerosis development can favor an excessive LPS-LP complex formation, and endotoxin presented in blood can inhibit lecithin:cholesterol acyltransferase (LCAT), one of the key enzymes of reverse cholesterol transport. Endotoxin-binding capacity of lipoproteins (LP) in patients with normolipidemia and hyperlipidemia types IIa and IV was estimated from label incorporation into different LP fractions isolated by means of sequential ultracentrifugation following serum preincubation with Salmonella minnesota R595 125I-labeled LPS. The effect of varied concentrations of S. minnesota R595 LPS on LCAT activity was evaluated from the overall esterifying activity of serum using [1,2-3H2]cholesterol-labeled substrate. The elevation of low density LP (LDL) and very low density LP (VLDL) contents in blood serum in hyperlipidemia types IIa and IV, respectively, resulted in significant elevation of LPS binding to these fractions. LPS added to the blood serum leads to the dose-dependent decrease in LCAT activity. The revealed phenomena of elevated LPS binding to atherogenic LP fractions in hypercholesterolemia and endotoxin-induced LCAT inhibition suggest the pathogenic role of LPS-LP complexes in atherogenesis.

Lecithin: cholesterol acyltransferase is insufficient to prevent oxidative modification of low-density lipoprotein.

We tried to confirm the antioxidative capability of lecithin:cholesterol acyltransferase (LCAT) reported by Vohl et al. [Biochemistry (1999) 38, 5976-5981]. The enzyme solution protected LDL against oxidation. However, this protection was not due to LCAT enzyme, but to some unknown low-molecular-weight substance(s) in the solution; LCAT itself exerted little protective effect against LDL oxidation.

Haptoglobin inhibits lecithin-cholesterol acyltransferase in human ovarian follicular fluid.

The activity of the enzyme lecithin-cholesterol acyltransferase (LCAT; E.C. 2.3.1.43) is involved in the removal of cholesterol excess from peripheral cells. This activity is stimulated by the HDL (high density lipoprotein) apolipoprotein A1 (ApoA1). Haptoglobin (Hpt) was previously found to be associated with ApoA1 in ovarian follicular fluid. LCAT activity was analyzed in follicular fluids, collected from an IVF program, containing different amounts of Hpt or Hpt/ApoA1 ratio. Addition of purified Hpt to follicular fluid caused a decrease in the enzyme activity, which was measured as the rate of synthesis of cholesteryl esters. In the fractions of fluid proteins, as obtained by gel filtration chromatography, Hpt and HDL were titrated by ELISA while the LCAT activity was assayed by using radioactive cholesterol and purified HDL. When isolated LCAT was incubated with fractions containing different Hpt/ApoA1 ratios, the enzyme activity was found negatively correlated with the Hpt/ApoA1 ratio (P < 0.01). LCAT kinetic parameters were measured in two fractions with the same amount of ApoA1 (5 microg/ml) but different amounts of Hpt (0.69 or 3.77 microg/ml): the V(max) did not change while the K(m) values were 24.1 or 78.6 microM in the presence of the low or high Hpt level, respectively. The analysis of fluids associated with cytoplasmically mature MII oocytes, in a cross-sectional study, confirmed that a negative correlation exists between the Hpt/ApoA1 ratio and the LCAT activity (P < 0.01). The results suggest that Hpt inhibits the reverse transport of cholesterol by preventing ApoA1 stimulation of the LCAT activity.

Ldl modified by hypochlorous acid is a potent inhibitor of lecithin-cholesterol acyltransferase activity.

Modification of low density lipoprotein (LDL) by myeloperoxidase-generated HOCl has been implicated in human atherosclerosis. Incubation of LDL with HOCl generates several reactive intermediates, primarily N-chloramines, which may react with other biomolecules. In this study, we investigated the effects of HOCl-modified LDL on the activity of lecithin-cholesterol acyltransferase (LCAT), an enzyme essential for high density lipoprotein maturation and the antiatherogenic reverse cholesterol transport pathway. We exposed human LDL (0.5 mg protein/mL) to physiological concentrations of HOCl (25 to 200 micromol/L) and characterized the resulting LDL modifications to apolipoprotein B and lipids; the modified LDL was subsequently incubated with apolipoprotein B-depleted plasma (density >1.063 g/mL fraction), which contains functional LCAT. Increasing concentrations of HOCl caused various modifications to LDL, primarily, loss of lysine residues and increases in N-chloramines and electrophoretic mobility, whereas lipid hydroperoxides were only minor products. LCAT activity was extremely sensitive to HOCl-modified LDL and was reduced by 23% and 93% by LDL preincubated with 25 and 100 micromol/L HOCl, respectively. Addition of 200 micromol/L ascorbate or N-acetyl derivatives of cysteine or methionine completely prevented LCAT inactivation by LDL preincubated with