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Cardiovascular disease is the leading cause of death in end-stage renal disease (ESRD) patients treated with hemodialysis. An important contributor might be a decline in the cardioprotective effects of high-density lipoprotein (HDL). One important factor affecting HDL's cardioprotective properties may involve the alterations of protein composition in HDL. In the current study, we used complementary proteomics approaches to detect and quantify relative levels of proteins in HDL isolated from control and ESRD subjects. Shotgun proteomics analysis of HDL isolated from 20 control and 40 ESRD subjects identified 63 proteins in HDL. Targeted quantitative proteomics by isotope-dilution selective reaction monitoring revealed that 22 proteins were significantly enriched and 6 proteins were significantly decreased in ESRD patients. Strikingly, six proteins implicated in renal disease, including B2M, CST3, and PTGDS, were markedly increased in HDL of uremic subjects. Moreover, several of these proteins (SAA1, apoC-III, PON1, etc.) have been associated with atherosclerosis. Our observations indicate that the HDL proteome is extensively remodeled in uremic subjects. Alterations of the protein cargo of HDL might impact HDL's proposed cardioprotective properties. Quantifying proteins in HDL may be useful in the assessment of cardiovascular risk in patients with ESRD and in assessing response to therapeutic interventions.

High-density lipoprotein (HDL) retards atherosclerosis by accepting cholesterol from the artery wall. However, the structure of the proposed acceptor, monomeric apolipoprotein A-I (apoA-I), the major protein of HDL, is poorly understood. Two published models for monomeric apoA-I used cross-linking distance constraints to derive best fit conformations. This approach has limitations. (i) Cross-linked peptides provide no information about secondary structure. (ii) A protein chain can be folded in multiple ways to create a best fit. (iii) Ad hoc folding of a secondary structure is unlikely to produce a stable orientation of hydrophobic and hydrophilic residues. To address these limitations, we used a different approach. We first noted that the dimeric apoA-I crystal structure, (Δ185-243)apoA-I, is topologically identical to a monomer in which helix 5 forms a helical hairpin, a monomer with a hydrophobic cleft running the length of the molecule. We then realized that a second crystal structure, (Δ1-43)apoA-I, contains a C-terminal structure that fits snuggly via aromatic and hydrophobic interactions into the hydrophobic cleft. Consequently, we combined these crystal structures into an initial model that was subjected to molecular dynamics simulations. We tested the initial and simulated models and the two previously published models in three ways: against two published data sets (domains predicted to be helical by H/D exchange and six spin-coupled residues) and against our own experimentally determined cross-linking distance constraints. We note that the best fit simulation model, superior by all tests to previously published models, has dynamic features of a molten globule with interesting implications for the functions of apoA-I.

The antiatherogenic properties of apolipoprotein A-I (apoA-I) are derived, in part, from lipidation-state-dependent structural elements that manifest at different stages of apoA-I's progression from lipid-free protein to spherical high-density lipoprotein (HDL). Previously, we reported the structure of apoA-I's N-terminus on reconstituted HDLs (rHDLs) of different sizes. We have now investigated at the single-residue level the conformational adaptations of three regions in the central domain of apoA-I (residues 119-124, 139-144, and 164-170) upon apoA-I lipid binding and HDL formation. An important function associated with these residues of apoA-I is the activation of lecithin:cholesterol acyltransferase (LCAT), the enzyme responsible for catalyzing HDL maturation. Structural examination was performed by site-directed tryptophan fluorescence and spin-label electron paramagnetic resonance spectroscopies for both the lipid-free protein and rHDL particles 7.8, 8.4, and 9.6 nm in diameter. The two methods provide complementary information about residue side chain mobility and molecular accessibility, as well as the polarity of the local environment at the targeted positions. The modulation of these biophysical parameters yielded new insight into the importance of structural elements in the central domain of apoA-I. In particular, we determined that the loosely lipid-associated structure of residues 134-145 is conserved in all rHDL particles. Truncation of this region completely abolished LCAT activation but did not significantly affect rHDL size, reaffirming the important role of this structural element in HDL function.

Phospholipid transfer protein (PLTP), which associates with apolipoprotein A-I (the major HDL protein), plays a key role in lipoprotein remodeling. Because its level in plasma increases during acute inflammation, it may also play previously unsuspected roles in the innate immune system. To gain further insight into its potential physiological functions, we isolated complexes containing PLTP from plasma by immunoaffinity chromatography and determined their composition. Shotgun proteomics revealed that only 6 of the 24 proteins detected in the complexes were apolipoproteins. The most abundant proteins were clusterin (apoJ), PLTP itself, coagulation factors, complement factors, and apoA-I. Remarkably, 20 of the 24 proteins had known protein-protein interactions. Biochemical studies confirmed two previously established interactions and identified five new ones between PLTP and proteins. Moreover, clusterin, apoA-I, and apoE preserved the lipid-transfer activity of recombinant PLTP in the absence of lipid, indicating that these interactions may have functional significance. Unexpectedly, lipids accounted for only 3% of the mass of the PLTP complexes. Collectively, our observations indicate that PLTP in human plasma resides on lipid-poor complexes dominated by clusterin and proteins implicated in host defense and inflammation. They further suggest that protein-protein interactions drive the formation of PLTP complexes in plasma.

Accumulation of low-density lipoprotein (LDL)-derived cholesterol by artery wall macrophages triggers atherosclerosis, the leading cause of cardiovascular disease. Conversely, high-density lipoprotein (HDL) retards atherosclerosis by promoting cholesterol efflux from macrophages by the membrane-associated ATP-binding cassette transporter A1 (ABCA1) pathway. HDL has been proposed to lose its cardioprotective effects in subjects with atherosclerosis, but the underlying mechanisms are poorly understood. One potential pathway involves oxidative damage by myeloperoxidase (MPO), a heme enzyme secreted by human artery wall macrophages. We used mass spectrometry to demonstrate that HDL isolated from patients with established cardiovascular disease contains elevated levels of 3-chlorotyrosine and 3-nitrotyrosine, two characteristic products of MPO. When apolipoprotein A-I (apoA-I), the major HDL protein, was oxidized by MPO, its ability to promote cellular cholesterol efflux by ABCA1 was impaired. Moreover, oxidized apoA-I was unable to activate lecithin:cholesterol acyltransferase (LCAT), which rapidly converts free cholesterol to cholesteryl ester, a critical step in HDL maturation. Biochemical studies implicated tyrosine chlorination and methionine oxygenation in the loss of ABCA1 and LCAT activity by oxidized apoA-I. Oxidation of specific residues in apoA-I inhibited two key steps in cholesterol efflux from macrophages, raising the possibility that MPO initiates a pathway for generating dysfunctional HDL in humans.

The pathophysiology of cystic fibrosis (CF) lung disease remains incompletely understood. Novel mechanisms in the pathogenesis of CF lung disease may be discovered by studying the patterns of protein expression in bronchoalveolar lavage fluid (BALF). We used shotgun proteomics to analyze BALF samples from 8 CF and 4 control subjects. Differential protein expression between CF and control subjects was determined using spectral counting and statistical analysis. Using Gene Ontology analysis, we identified enriched biological modules and then applied network analysis to construct a protein interaction map in CF lung disease. Shotgun proteomics analysis of BALF identified hundreds of proteins whose differential enrichment or depletion robustly distinguished the CF phenotype from normal controls. Functional categorization and network analysis identified key processes, including the immune response and proteolytic activity that are known contributors to CF lung disease. Importantly, this approach also implicated abnormalities in previously unsuspected pathways, such as dysregulation of the complement system that may have critical roles in the pathogenesis of CF lung disease. By integrating shotgun proteomics with statistical and computational analyses, we have developed a promising approach to understand the pathophysiology of CF lung disease. Our approach should be applicable to a wide range of proteomics-based clinical research.

High-density lipoprotein (HDL) mediates reverse cholesterol transport (RCT), wherein excess cholesterol is conveyed from peripheral tissues to the liver and steroidogenic organs. During this process HDL continually transitions between subclass sizes, each with unique biological activities. For instance, RCT is initiated by the interaction of lipid-free/lipid-poor apolipoprotein A-I (apoA-I) with ABCA1, a membrane-associated lipid transporter, to form nascent HDL. Because nearly all circulating apoA-I is lipid-bound, the source of lipid-free/lipid-poor apoA-I is unclear. Lecithin:cholesterol acyltransferase (LCAT) then drives the conversion of nascent HDL to spherical HDL by catalyzing cholesterol esterification, an essential step in RCT. To investigate the relationship between HDL particle size and events critical to RCT such as LCAT activation and lipid-free apoA-I production for ABCA1 interaction, we reconstituted five subclasses of HDL particles (rHDL of 7.8, 8.4, 9.6, 12.2, and 17.0 nm in diameter, respectively) using various molar ratios of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, free cholesterol, and apoA-I. Kinetic analyses of this comprehensive array of rHDL particles suggest that apoA-I stoichiometry in rHDL is a critical factor governing LCAT activation. Electron microscopy revealed specific morphological differences in the HDL subclasses that may affect functionality. Furthermore, stability measurements demonstrated that the previously uncharacterized 8.4 nm rHDL particles rapidly convert to 7.8 nm particles, concomitant with the dissociation of lipid-free/lipid-poor apoA-I. Thus, lipid-free/lipid-poor apoA-I generated by the remodeling of HDL may be an essential intermediate in RCT and HDL's in vivo maturation.

Detecting differentially expressed proteins is a key goal of proteomics. We describe a label-free method, the spectral index, for analyzing relative protein abundance in large-scale data sets derived from biological samples by shotgun proteomics. The spectral index is comprised of two biochemically plausible features: relative protein abundance (assessed by spectral counts) and the number of samples within a group with detectable peptides. We combined the spectral index with permutation analysis to establish confidence intervals for assessing differential protein expression in bronchoalveolar lavage fluid from cystic fibrosis and control subjects. Significant differences in protein abundance determined by the spectral index agreed well with independent biochemical measurements. When used to analyze simulated data sets, the spectral index outperformed four other statistical tests (Student's t-test, G-test, Bayesian t-test, and Significance Analysis of Microarrays) by correctly identifying the largest number of differentially expressed proteins. Correspondence analysis and functional annotation analysis indicated that the spectral index improves the identification of enriched proteins corresponding to clinical phenotypes. The spectral index is easily implemented and statistically robust, and its results are readily interpreted graphically. Therefore, it should be useful for biomarker discovery and comparisons of protein expression between normal and disease states.

Most studies of protein oxidation have typically focused on the reactivity of single amino acid side chains while ignoring the potential importance of adjacent sequences in directing the reaction pathway. We previously showed that hypochlorous acid (HOCl), a specific product of myeloperoxidase, inactivates matrilysin by modifying adjacent tryptophan and glycine (WG) residues in the catalytic domain. Here, we use model peptides that mimic the region of matrilysin involved in this reaction, VVWGTA, VVWATA, and the library VVWXTA, to determine whether specific sequence motifs are targeted for chlorination or oxygenation by myeloperoxidase. Our results demonstrate that HOCl generated by myeloperoxidase or activated neutrophils converts the peptide VVWGTA to a chlorinated product, WG+32(Cl). Tandem mass spectrometry in concert with high resolution 1H and two-dimensional NMR analysis revealed that the modification required cross-linking of the tryptophan to the amide of glycine followed by chlorination of the indole ring of tryptophan. In contrast, when glycine in the peptide was replaced with alanine, the major products were mono- and dioxygenated tryptophan residues. When the peptide library VVWXTA (where X represents all 20 common amino acids) was exposed to HOCl, only WG produced a high yield of the chloroindolenine derivative. However, when glycine was replaced by other amino acids, oxygenated tryptophan derivatives were the major products. Our observations indicate that WG may represent a specific sequence motif in proteins that is targeted for chlorination by myeloperoxidase.

Oxidized low-density lipoprotein (LDL) is implicated in atherogenesis, and human atherosclerotic lesions contain LDL oxidized by myeloperoxidase, a heme protein secreted by activated phagocytes. Using hydrogen peroxide (H(2)O(2)), myeloperoxidase generates hypochlorous acid (HOCl), a powerful oxidant. We now demonstrate that HOCl produces sulfenamides, sulfinamides, and sulfonamides in model peptides, which suggests a potential mechanism for LDL oxidation and cross-linking. When we exposed the synthetic peptide PFKCG to HOCl, the peptide's thiol residue reacted rapidly, generating a near-quantitative yield of products. Tandem mass spectrometric analysis identified the products as the sulfenamide, sulfinamide, and sulfonamide, all formed by intramolecular cross-linking of the peptide's thiol and lysine residues. An intramolecular sulfinamide was also observed after the peptide PFRCG was exposed to HOCl, indicating that the guanidine group of arginine can also form a sulfur-nitrogen cross-link. The synthetic peptide PFVCG, which contains a free thiol residue but lacks nucleophilic amino acid side chains, formed an intermolecular sulfonamide when exposed to HOCl. Tandem mass spectrometric analysis of the dimer revealed that the free N-terminal amino group of one PFVCG molecule cross-linked with the thiol residue of another. This peptide also formed intermolecular sulfonamide cross-links with N(alpha)-acetyllysine after exposure to HOCl, demonstrating that the epsilon-amino group of a lysine residue can undergo a similar reaction. Moreover, human neutrophils used the myeloperoxidase-H(2)O(2) system to generate sulfinamides in model peptides containing lysine or arginine residues. Collectively, our observations raise the possibility that HOCl generated by myeloperoxidase contributes to intramolecular and intermolecular protein cross-linking in the artery wall. Myeloperoxidase might also use this mechanism to form sulfur-nitrogen cross-links in other inflammatory conditions.

Phagocytic oxidants have been implicated in tissue injury and oncogenesis, and their pathophysiological role in modifying nucleobases and amino acids has been widely explored. Their ability to cross-link proteins and DNA, however, has not been considered, even though reversible DNA-protein interactions are key to gene expression and to DNA replication and repair. In the current studies, we show that hypochlorous acid (HOCl), generated by the myeloperoxidase-hydrogen peroxide-chloride system of phagocytes, cross-links single-stranded DNA-binding protein (SSB) to single-stranded oligonucleotides. Exposure of SSB and a homopolymer of radiolabeled thymidine (dT(40)) to HOCl resulted in the formation of a radiolabeled band with slower mobility than the free oligonucleotide, as determined by denaturing polyacrylamide gel electrophoresis. This radiolabeled band did not appear if the reaction mixture was treated with protease or nuclease, indicating that it represents a covalent complex of DNA and protein. Oligonucleotides of adenosine and cytidine behaved similarly to the thymidine oligonucleotide, demonstrating that they are also capable of participating in the cross-linking reaction. The covalent complex of radiolabeled dT(40) and SSB was also generated by chloramines and the complete myeloperoxidase-hydrogen peroxide-chloride system. The enzymatic reaction required each component of the system and was inhibited by heme poisons and chloride-free conditions, implicating myeloperoxidase and HOCl. DNA-protein cross-links were generated in Escherichia coli exposed to HOCl, suggesting that double-stranded DNA is also a target for the reaction. These results indicate that long-lived chloramines and HOCl generated by myeloperoxidase can generate covalent DNA-protein cross-links that may contribute to the mutagenic and cytotoxic effects of phagocytes on microbial pathogens and host tissue.

Eosinophils use eosinophil peroxidase, hydrogen peroxide (H(2)O(2)), and bromide ion (Br(-)) to generate hypobromous acid (HOBr), a brominating intermediate. This potent oxidant may play a role in host defenses against invading parasites and eosinophil-mediated tissue damage. In this study, we explore the possibility that HOBr generated by eosinophil peroxidase might oxidize nucleic acids. When we exposed uracil, uridine, or deoxyuridine to reagent HOBr, each reaction mixture yielded a single major oxidation product that comigrated on reversed-phase HPLC with the corresponding authentic brominated pyrimidine. The eosinophil peroxidase-H(2)O(2)-Br(-) system also converted uracil into a single major oxidation product, and the yield was near-quantitative. Mass spectrometry, HPLC, UV--visible spectroscopy, and NMR spectroscopy identified the product as 5-bromouracil. Eosinophil peroxidase required H(2)O(2) and Br(-) to produce 5-bromouracil, implicating HOBr as an intermediate in the reaction. Primary and secondary bromamines also brominated uracil, suggesting that long-lived bromamines also might be physiologically relevant brominating intermediates. Human eosinophils used the eosinophil peroxidase-H(2)O(2)-Br(-) system to oxidize uracil. The product was identified as 5-bromouracil by mass spectrometry, HPLC, and UV--visible spectroscopy. Collectively, these results indicate that HOBr generated by eosinophil peroxidase oxidizes uracil to 5-bromouracil. Thymidine phosphorylase, a pyrimidine salvage enzyme, transforms 5-bromouracil to 5-bromodeoxyridine, a mutagenic analogue of thymidine. These findings raise the possibility that halogenated nucleobases generated by eosinophil peroxidase exert cytotoxic and mutagenic effects at eosinophil-rich sites of inflammation.

Reactive intermediates generated by phagocytes damage DNA and may contribute to the link between chronic inflammation and cancer. Myeloperoxidase, a heme protein secreted by activated phagocytes, is a potential catalyst for such reactions. Recent studies demonstrate that this enzyme uses hydrogen peroxide (H2O2) and nitrite (NO2-) to generate reactive nitrogen species which convert tyrosine to 3-nitrotyrosine. We now report that activated human neutrophils use myeloperoxidase, H2O2, and NO2- to nitrate 2'-deoxyguanosine, one of the nucleosides of DNA. Through HPLC, UV/vis spectroscopy, and mass spectrometry, the two major products of this reaction were identified as 8-nitroguanine and 8-nitro-2'-deoxyguanosine. Nitration required each component of the complete enzymatic system and was inhibited by catalase and heme poisons. However, it was independent of chloride ion and little affected by scavengers of hypochlorous acid, suggesting that the reactive agent is a nitrogen dioxide-like species that results from the one-electron oxidation of NO2- by myeloperoxidase. Alternatively, 2'-deoxyguanosine might be oxidized directly by the enzyme to yield a radical species which subsequently reacts with NO2- or NO2* to generate the observed products. Human neutrophils stimulated with phorbol ester also generated 8-nitroguanine and 8-nitro-2'-deoxyguanosine. The reaction required NO2- and was inhibited by catalase and heme poisons, implicating myeloperoxidase in the cell-mediated pathway. These results indicate that human neutrophils use the myeloperoxidase-H2O2-NO2- system to generate reactive species that can nitrate the C-8 position of 2'-deoxyguanosine. Our observations raise the possibility that reactive nitrogen species generated by myeloperoxidase and other peroxidases contribute to nucleobase oxidation and tissue injury at sites of inflammation.

The adducts that form when aldehydes modify proteins have been implicated in the pathogenesis of vascular disease and aging. Our previous studies indicated that p-hydroxyphenylacetaldehyde (pHA), the major product of L-tyrosine oxidation by the myeloperoxidase/hydrogen peroxide/chloride system of phagocytes, covalently modifies the epsilon-amino group of lysine residues at sites of inflammation. Here, we report that pHA also reacts with the amino group of synthetic phospholipids and red blood cell model systems. Using fast atom bombardment mass spectrometric analysis of ethanolamine glycerophospholipid or serine glycerophospholipid incubated with pHA and NaBH3CN, we detected products that were consistent with reduced phospholipid Schiff base adducts. We confirmed the reaction of the aldehyde with the amino group through 1H NMR and mass spectrometric analysis of polar headgroups recovered from the modified and reduced parent lipid. When phospholipid model systems and cell membranes were exposed to physiological levels of L-tyrosine and the myeloperoxidase/hydrogen peroxide/chloride system followed by treatment with NaBH3CN, reduced Schiff base adducts of pHA with ethanolamine glycerophospholipid and serine glycerophospholipid (pHA-PE and pHA-PS, respectively) were produced. The reaction required myeloperoxidase, hydrogen peroxide, L-tyrosine, and chloride ion; it was inhibited by catalase or heme poisons, implicating hydrogen peroxide and peroxidase in the pathway. Collectively, these results demonstrate that an aldehyde generated by the myeloperoxidase system of phagocytes can covalently modify the amino groups of phosphatidylethanolamine and phosphatidylserine. Because amino glycerophospholipids are critical components of cell membranes and circulating lipoproteins such as LDL, similar reactions may play important roles in the initiation or progression of disease at sites of inflammation.

We have recently demonstrated that activated phagocytes employ the heme protein myeloperoxidase, H2O2, and Cl- to oxidize the aromatic amino acid l-tyrosine to the reactive aldehyde p-hydroxyphenylacetaldehyde. We now present evidence for the generality of this reaction by demonstrating that neutrophils employ the myeloperoxidase-H2O2-Cl- system to oxidize nearly all of the common alpha-amino acids to yield a family of reactive aldehydes. Chemical characterization suggested that reactive carbonyl moieties were generated during amino acid oxidation by myeloperoxidase. The structures of amino-acid-derived aldehydes were confirmed using a variety of mass spectrometric methods. Aldehyde production required myeloperoxidase, H2O2, Cl-, and an amino acid; it was inhibited by heme poisons and catalase. Hypochlorous acid was the apparent oxidizing intermediate because its addition to alpha-amino acids resulted in the formation of the anticipated aldehyde. Stimulated human neutrophils likewise generated aldehydes from all classes of alpha-amino acids by a pathway inhibited by heme poisons and catalase, implicating myeloperoxidase and H2O2 in the cell-mediated reaction. Aldehyde production accounted for a significant fraction of the H2O2 generated by stimulated neutrophils at physiological concentrations of amino acids. Collectively, these results suggest that amino-acid-derived aldehydes represent a product of reactive oxidant species generated by activated phagocytes.

It is believed that HDL protects against atherosclerosis by removing excess cholesteryl esters from cells of the artery wall. Previous studies have suggested that HDL depletes cells of cholesteryl esters both by stimulating cholesterol efflux from the plasma membrane and by activating transport processes that divert cholesterol from the cholesteryl ester cycle, but it is unknown if these are independent processes. We previously found that HDL oxidized by tyrosyl radical has a markedly enhanced ability to promote the removal of cholesterol from cultured cells [Francis, G. A., et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 6631-6635]. Here we show that incubation of cholesterol-loaded human fibroblasts with low concentrations of tyrosylated HDL depleted cells of cholesteryl esters and increased cellular free cholesterol without increasing efflux of cholesterol into the medium as compared to incubation with untreated HDL. Cells preincubated with tyrosylated HDL and then exposed to a variety of cholesterol acceptors exhibited significantly higher rates of free cholesterol efflux than did cells preincubated with HDL. This effect was observed in the presence or absence of an inhibitor of acyl CoA:cholesterol acyltransferase (ACAT) and was independent of cholesteryl ester hydrolysis, suggesting that alterations in cholesteryl ester cycle enzymes were not responsible for the loss of cholesteryl esters. In contrast to the reduction of cholesteryl esters, the rates of cholesterol and phospholipid efflux from the plasma membranes of cells exposed to tyrosylated HDL and HDL were identical. These results suggest for the first time that a mechanism exists to deplete cellular cholesteryl esters and the cholesterol substrate pool for esterification by ACAT prior to the removal of cholesterol from the plasma membrane. Identification of products in tyrosylated HDL responsible for this redistribution of cellular cholesterol may provide important insights into mechanisms of intracellular cholesterol trafficking and the ability of modified forms of HDL to protect the artery against wall pathological cholesterol accumulation.

Myeloperoxidase, a heme protein secreted by activated phagocytes, uses hydrogen peroxide to produce potent cytotoxins. One important substrate is chloride, which is converted to hypochlorous acid (HOCl). This diffusible oxidant plays a critical role in the destruction of invading pathogens. Under pathological conditions, HOCl may also injure normal tissue. Recent studies have shown that myeloperoxidase is a component of human atherosclerotic lesions. Because oxidized lipoproteins may play a central role in atherogenesis, we have explored the possibility that cholesterol is a target for damage by myeloperoxidase. Three major classes of sterol oxidation products were apparent when cholesterol-phosphatidylcholine multilamellar vesicles which had been exposed to a myeloperoxidase-hydrogen peroxide-chloride system were subsequently analyzed by normal-phase thin layer chromatography. The products were identified by gas chromatography-mass spectrometry as cholesterol alpha- and beta-chlorohydrins (6 beta-chlorocholestane-3 beta,5 alpha-diol and 5 alpha-chlorocholestane-3 beta,6 beta-diol), cholesterol alpha- and beta-epoxides (cholesterol 5 alpha,6 alpha-epoxide and cholesterol 5 beta,6 beta-epoxide), and a novel cholesterol chlorohydrin. Conversion of cholesterol to the oxidation products required active myeloperoxidase, hydrogen peroxide, and halide and could be blocked by catalase or by scavengers of HOCl. Moreover, in the absence of the enzymatic system, reagent HOCl generated the same distribution of products. These results indicate that myeloperoxidase can convert cholesterol to chlorohydrins and epoxides by a reaction involving HOCl. Other oxygenated sterols are cytotoxic and mutagenic and are potent regulators of cholesterol homeostasis in cultured mammalian cells. Cholesterol chlorohydrins might similarly mediate powerful biological effects in the artery wall. Because chlorohydrins are stable under our experimental conditions, chlorinated sterols may prove useful as markers for lipoproteins oxidatively damaged by activated phagocytes.