Structure of complex III with bound antimalarial agent CK-2-68 provides insights into selective inhibition of Plasmodium cytochrome bc1 complexes.

Among the various components of the protozoan Plasmodium mitochondrial respiratory chain, only Complex III is a validated cellular target for antimalarial drugs. The compound CK-2-68 was developed to specifically target the alternate NADH dehydrogenase of the malaria parasite respiratory chain, but the true target for its antimalarial activity has been controversial. Here, we report the cryo-EM structure of mammalian mitochondrial Complex III bound with CK-2-68 and examine the structure-function relationships of the inhibitor's selective action on Plasmodium. We show that CK-2-68 binds specifically to the quinol oxidation site of Complex III, arresting the motion of the iron-sulfur protein subunit, which suggests an inhibition mechanism similar to that of Pf-type Complex III inhibitors such as atovaquone, stigmatellin, and UHDBT. Our results shed light on the mechanisms of observed resistance conferred by mutations, elucidate the molecular basis of the wide therapeutic window of CK-2-68 for selective action of Plasmodium vs. host cytochrome bc1, and provide guidance for future development of antimalarials targeting Complex III.

Crystal structure of bacterial cytochrome bc1 in complex with azoxystrobin reveals a conformational switch of the Rieske iron-sulfur protein subunit.

Cytochrome bc1 complexes (cyt bc1), also known as complex III in mitochondria, are components of the cellular respiratory chain and of the photosynthetic apparatus of non-oxygenic photosynthetic bacteria. They catalyze electron transfer (ET) from ubiquinol to cytochrome c and concomitantly translocate protons across the membrane, contributing to the cross-membrane potential essential for a myriad of cellular activities. This ET-coupled proton translocation reaction requires a gating mechanism that ensures bifurcated electron flow. Here, we report the observation of the Rieske iron-sulfur protein (ISP) in a mobile state, as revealed by the crystal structure of cyt bc1 from the photosynthetic bacterium Rhodobacter sphaeroides in complex with the fungicide azoxystrobin. Unlike cyt bc1 inhibitors stigmatellin and famoxadone that immobilize the ISP, azoxystrobin causes the ISP-ED to separate from the cyt b subunit and to remain in a mobile state. Analysis of anomalous scattering signals from the iron-sulfur cluster of the ISP suggests the existence of a trajectory for electron delivery. This work supports and solidifies the hypothesis that the bimodal conformation switch of the ISP provides a gating mechanism for bifurcated ET, which is essential to the Q-cycle mechanism of cyt bc1 function.

Hydrogen Bonding to the Substrate Is Not Required for Rieske Iron-Sulfur Protein Docking to the Quinol Oxidation Site of Complex III.

Complex III or the cytochrome (cyt) bc1 complex constitutes an integral part of the respiratory chain of most aerobic organisms and of the photosynthetic apparatus of anoxygenic purple bacteria. The function of cyt bc1 is to couple the reaction of electron transfer from ubiquinol to cytochrome c to proton pumping across the membrane. Mechanistically, the electron transfer reaction requires docking of its Rieske iron-sulfur protein (ISP) subunit to the quinol oxidation site (QP) of the complex. Formation of an H-bond between the ISP and the bound substrate was proposed to mediate the docking. Here we show that the binding of oxazolidinedione-type inhibitors famoxadone, jg144, and fenamidone induces docking of the ISP to the QP site in the absence of the H-bond formation both in mitochondrial and bacterial cyt bc1 complexes, demonstrating that ISP docking is independent of the proposed direct ISP-inhibitor interaction. The binding of oxazolidinedione-type inhibitors to cyt bc1 of different species reveals a toxophore that appears to interact optimally with residues in the QP site. The effect of modifications or additions to the toxophore on the binding to cyt bc1 from different species could not be predicted from structure-based sequence alignments, as demonstrated by the altered binding mode of famoxadone to bacterial cyt bc1.

Universal Stress Protein Regulates Electron Transfer and Superoxide Generation Activities of the Cytochrome bc1 Complex from Rhodobacter sphaeroides.

Interactions between Rhodobacter sphaeroides cytochrome bc1 complex (Rsbc1) and soluble cytosolic proteins were studied by a precipitation pull-down technique. After being purified, detergent-dispersed Rsbc1 complex was incubated with soluble cytosolic fraction and then dialyzed in the absence of detergent; the interacting proteins were coprecipitated with Rsbc1 complex upon centrifugation. One of the cytosolic proteins pulled down by Rsbc1 complex was identified by liquid chromatography-coupled tandem mass spectrometry (LC/MS/MS) to be the reported R. sphaeroides universal stress protein (UspA). Incubating purified UspA with the detergent dispersed bc1 complex resulted in an increase in the Rsbc1 complex activity by 60% and a decrease in superoxide generation activity by the complex by more than 70%. These UspA effects were only observed with Rsbc1 complexes containing subunit IV and assayed under aerobic conditions. These results suggest that the interaction between UspA and Rsbc1 complex may play an important role in R. sphaeroides cells during oxidative stress. Using a biotin label transfer technique, cytochrome c1 of the Rsbc1 complex was identified as the interacting site for UspA.

Effect of mutations of arginine 94 on proton pumping, electron transfer, and superoxide anion generation in cytochrome b of the bc1 complex from Rhodobacter sphaeroides.

Proton transfer involving internal water molecules that provide hydrogen bonds and facilitate proton diffusion has been identified in some membrane proteins. Arg-94 in cytochrome b of the Rhodobacter sphaeroides bc(1) complex is fully conserved and is hydrogen-bonded to the heme propionate and a chain of water molecules. To further elucidate the role of Arg-94, we generated the mutations R94A, R94D, and R94N. The wild-type and mutant bc(1) complexes were purified and then characterized. The results show that substitution of Arg-94 decreased electron transfer activity and proton pumping capability and increased O(2)(.) production, suggesting the importance of Arg-94 in the catalytic mechanism of the bc(1) complex in R. sphaeroides. This also suggests that the transport of H(+), O(2), and O(2)(.) in the bc(1) complex may occur by the same pathway.

Structural analysis of cytochrome bc1 complexes: implications to the mechanism of function.

The cytochrome bc1 complex (bc1) is the mid-segment of the cellular respiratory chain of mitochondria and many aerobic prokaryotic organisms; it is also part of the photosynthetic apparatus of non-oxygenic purple bacteria. The bc1 complex catalyzes the reaction of transferring electrons from the low potential substrate ubiquinol to high potential cytochrome c. Concomitantly, bc1 translocates protons across the membrane, contributing to the proton-motive force essential for a variety of cellular activities such as ATP synthesis. Structural investigations of bc1 have been exceedingly successful, yielding atomic resolution structures of bc1 from various organisms and trapped in different reaction intermediates. These structures have confirmed and unified results of decades of experiments and have contributed to our understanding of the mechanism of bc1 functions as well as its inactivation by respiratory inhibitors. This article is part of a Special Issue entitled: Respiratory complex III and related bc complexes.

Generation, characterization and crystallization of a cytochrome c(1)-subunit IV fused cytochrome bc(1) complex from Rhodobacter sphaeroides.

Cytochrome bc(1) complex catalyzes the reaction of electron transfer from ubiquinol to cytochrome c (or cytochrome c(2)) and couples this reaction to proton translocation across the membrane. Crystallization of the Rhodobacter sphaeroides bc(1) complex resulted in crystals containing only three core subunits. To mitigate the problem of subunit IV being dissociated from the three-subunit core complex during crystallization, we recently engineered an R. sphaeroides mutant in which the N-terminus of subunit IV was fused to the C-terminus of cytochrome c(1) with a 14-glycine linker between the two fusing subunits, and a 6-histidine tag at the C-terminus of subunit IV (c(1)-14Gly-IV-6His). The purified fusion mutant complex shows higher electron transfer activity, more structural stability, and less superoxide generation as compared to the wild-type enzyme. Preliminary crystallization attempts with this mutant complex yielded crystals containing four subunits and diffracting X-rays to 5.5Å resolution.

Oxygen dependent electron transfer in the cytochrome bc(1) complex.

The effect of molecular oxygen on the electron transfer activity of the cytochrome bc(1) complex was investigated by determining the activity of the complex under the aerobic and anaerobic conditions. Molecular oxygen increases the activity of Rhodobacter sphaeroides bc(1) complex up to 82%, depending on the intactness of the complex. Since oxygen enhances the reduction rate of heme b(L), but shows no effect on the reduction rate of heme b(H), the effect of oxygen in the electron transfer sequence of the cytochrome bc(1) complex is at the step of heme b(L) reduction during bifurcated oxidation of ubiquinol.

ADAMTS-7 expression increases in the early stage of angiotensin II-induced renal injury in elderly mice.

BACKGROUND/AIMS: We investigated the recently described family of proteinases, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTs), and matrix metalloproteinases (MMPs) as inflammatory mediators in inflammatory kidney damage by studying ADAMTS-1, -4, and -7 and MMP-9 expression in elderly mouse kidneys after angiotensin II (Ang II) administration. METHODS: Ang II (2.5 µg/kg/min) or norepinephrine (8.3 µg/kg/min) was subcutaneously infused in old mice. Renal injury was assessed by hematoxylin-eosin staining, 24-h albuminuria, and immunohistochemistry to evaluate inflammatory cell markers. The mRNA and protein expression of ADAMTS-1, -4, and -7 and MMP-9 were determined using real-time PCR, Western blot, and immunohistochemistry 3 days after Ang II or norepinephrine administration. RESULTS: Elderly mice in the Ang II group developed hypertension and pathological kidney damage. The mRNA and protein levels of ADAMTS-7 in the Ang II group were 3.3 ± 1.1 (P = 0.019) and 1.6 ± 0.1 (P = 0.047) vs. 1.0 ± 0.1 and 1.0 ± 0.1 in the control group on day 3. In contrast, treatment with the hypertensive agent norepinephrine did not lead to obvious renal damage or an increase in renal ADAMTS-7 expression. CONCLUSIONS: Renal ADAMTS-7 expression was induced by Ang II in elderly mice. The overexpression of ADATMTS-7 might contribute to early inflammatory kidney damage associated with aging.

Photoinduced electron transfer in cytochrome bc1: Dynamics of rotation of the Iron-sulfur protein during bifurcated electron transfer from ubiquinol to cytochrome c1 and cytochrome bL.

The electron transfer reactions within wild-type Rhodobacter sphaeroides cytochrome bc1 (cyt bc1) were studied using a binuclear ruthenium complex to rapidly photooxidize cyt c1. When cyt c1, the iron­sulfur center Fe2S2, and cyt bH were reduced before the reaction, photooxidation of cyt c1 led to electron transfer from Fe2S2 to cyt c1 with a rate constant of ka = 80,000 s-1, followed by bifurcated reduction of both Fe2S2 and cyt bL by QH2 in the Qo site with a rate constant of k2 = 3000 s-1. The resulting Q then traveled from the Qo site to the Qi site and oxidized one equivalent each of cyt bL and cyt bH with a rate constant of k3 = 340 s-1. The rate constant ka was decreased in a nonlinear fashion by a factor of 53 as the viscosity was increased to 13.7. A mechanism that is consistent with the effect of viscosity involves rotational diffusion of the iron­sulfur protein from the b state with reduced Fe2S2 close to cyt bL to one or more intermediate states, followed by rotation to the final c1 state with Fe2S2 close to cyt c1, and rapid electron transfer to cyt c1.

Pulmonary rehabilitation restores limb muscle mitochondria and improves the intramuscular metabolic profile.

BACKGROUND: Exercise, as the cornerstone of pulmonary rehabilitation, is recommended to chronic obstructive pulmonary disease (COPD) patients. The underlying molecular basis and metabolic process were not fully elucidated. METHODS: Sprague-Dawley rats were classified into five groups: non-COPD/rest ( n  = 8), non-COPD/exercise ( n  = 7), COPD/rest ( n  = 7), COPD/medium exercise ( n  = 10), and COPD/intensive exercise ( n  = 10). COPD animals were exposed to cigarette smoke and lipopolysaccharide instillation for 90 days, while the non-COPD control animals were exposed to room air. Non-COPD/exercise and COPD/medium exercise animals were trained on a treadmill at a decline of 5° and a speed of 15 m/min while animals in the COPD/intensive exercise group were trained at a decline of 5° and a speed of 18 m/min. After eight weeks of exercise/rest, we used ultrasonography, immunohistochemistry, transmission electron microscopy, oxidative capacity of mitochondria, airflow-assisted desorption electrospray ionization-mass spectrometry imaging (AFADESI-MSI), and transcriptomics analyses to assess rectal femoris (RF). RESULTS: At the end of 90 days, COPD rats' weight gain was smaller than control by 59.48 ±â€Š15.33 g ( P  = 0.0005). The oxidative muscle fibers proportion was lower ( P  < 0.0001). At the end of additional eight weeks of exercise/rest, compared to COPD/rest, COPD/medium exercise group showed advantages in weight gain, femoral artery peak flow velocity (Δ58.22 mm/s, 95% CI: 13.85-102.60 mm/s, P  = 0.0104), RF diameters (Δ0.16 mm, 95% CI: 0.04-0.28 mm, P  = 0.0093), myofibrils diameter (Δ0.06 µm, 95% CI: 0.02-0.10 µm, P  = 0.006), oxidative muscle fiber percentage (Δ4.84%, 95% CI: 0.15-9.53%, P  = 0.0434), mitochondria oxidative phosphorylate capacity ( P  < 0.0001). Biomolecules spatial distribution in situ and bioinformatic analyses of transcriptomics suggested COPD-related alteration in metabolites and gene expression, which can be impacted by exercise. CONCLUSION: COPD rat model had multi-level structure and function impairment, which can be mitigated by exercise.

Cross-talk between mitochondrial malate dehydrogenase and the cytochrome bc1 complex.

The interactions between the mitochondrial cytochrome bc(1) complex and matrix-soluble proteins were studied by a precipitation pulldown technique. Purified, detergent-dispersed bc(1) complex was incubated with mitochondrial matrix proteins followed by dialysis in the absence of detergent. The interacting protein(s) was co-precipitated with bc(1) complex upon centrifugation. One of the matrix proteins pulled down by bc(1) complex was identified as mitochondrial malate dehydrogenase (MDH) by matrix-assisted laser desorption ionization time-of-flight mass spectrometry and confirmed by Western blotting with anti-MDH antibody. Using a cross-linking technique, subunits I, II (core I and II), and V of the bc(1) complex were identified as the interacting sites for MDH. Incubating purified MDH with the detergent dispersed bc(1) complex results in an increase of the activities of both the bc(1) complex and MDH. The effect of the bc(1) complex on the activities of MDH is unidirectional (oxaloacetate --> malate). These results suggest that the novel cross-talk between citric acid cycle enzymes and electron transfer chain complexes might play a regulatory role in mitochondrial bioenergetics.

Reaction mechanism of superoxide generation during ubiquinol oxidation by the cytochrome bc1 complex.

In addition to its main functions of electron transfer and proton translocation, the cytochrome bc(1) complex (bc(1)) also catalyzes superoxide anion (O(2)(*)) generation upon oxidation of ubiquinol in the presence of molecular oxygen. The reaction mechanism of superoxide generation by bc(1) remains elusive. The maximum O(2)(*) generation activity is observed when the complex is inhibited by antimycin A or inactivated by heat treatment or proteinase K digestion. The fact that the cytochrome bc(1) complex with less structural integrity has higher O(2)(*)-generating activity encouraged us to speculate that O(2)(*) is generated inside the complex, perhaps in the hydrophobic environment of the Q(P) pocket through bifurcated oxidation of ubiquinol by transferring its two electrons to a high potential electron acceptor, iron-sulfur cluster, and a low potential heme b(L) or molecular oxygen. If this speculation is correct, then one should see more O(2)(*) generation upon oxidation of ubiquinol by a high potential oxidant, such as cytochrome c or ferricyanide, in the presence of phospholipid vesicles or detergent micelles than in the hydrophilic conditions, and this is indeed the case. The protein subunits, at least those surrounding the Q(P) pocket, may play a role either in preventing the release of O(2)(*) from its production site to aqueous environments or in preventing O(2) from getting access to the hydrophobic Q(P) pocket and might not directly participate in superoxide production.

Functional genetic screening reveals the role of mitochondrial cytochrome b as a mediator of FAS-induced apoptosis.

Functional selection of genetic suppressor elements (GSEs), engineered gene fragments that interfere with the function of a particular gene product, was used to identify regulators of FAS-induced apoptosis. Chicken DF-1 cells expressing human FAS receptor and susceptible to FAS-induced apoptosis were infected with a GSE library consisting of randomly fragmented normalized chicken cDNAs in a replication-competent avian retroviral vector. Virus-producing cells were subjected to several rounds of selection using FAS agonistic antibodies, resulting in isolation of a set of GSEs conferring resistance to FAS-induced apoptosis. Surprisingly, one of the isolated GSEs encoded a 42 amino acid-long polypeptide derived from the C-terminal half of cytochrome b (Cyt b) encoded by the mitochondrial genome. Subsequent experiments showed that caspase 8-dependent cleavage of mitochondrial Cyt b and translocation of its C-terminal half into the cytoplasm occurred during FAS-induced apoptosis in both chicken and human cells. Ectopic cytoplasmic expression of either full-length Cyt b or its C-terminal half in several human cell lines induced apoptosis, which could be suppressed by the isolated GSE, but not by Bcl2 over-expression or Apaf-1 or cytochrome c knock-down. These results reveal a cytochrome c-independent branch of FAS-induced apoptosis involving cleavage and cytoplasmic release of mitochondrial Cyt b.

Effect of subunit IV on superoxide generation by Rhodobacter sphaeroides cytochrome bc(1) complex.

Previous studies indicate that the three-subunit cytochrome bc(1) core complex of Rhodobacter sphaeroides contains a fraction of the electron transfer activity of the wild-type enzyme. Addition of subunit IV to the core complex increases electron transfer activity to the same level as that of the wild-type complex. This activity increase may result from subunit IV preventing electron leakage, from the low potential electron transfer chain, and reaction with molecular oxygen, producing superoxide anion. This suggestion is based on the following observations: (1) the extent of cytochrome b reduction in the three-subunit core complex, by ubiquinol, in the presence of antimycin A, never reaches the same level as that in the wild-type complex; (2) the core complex produces 4 times as much superoxide anion as does the wild-type complex; and (3) when the core complex is reconstituted with subunit IVs having varying reconstitutive activities, the activity increase in reconstituted complexes correlates with superoxide production decrease and extent of cytochrome b reduction increase.

[Ischemia preconditioning attenuated myocardial ischemia via upregulating the expression of adiponectin in rat].

OBJECTIVE: To investigate whether adiponectin plays a role in the protection of myocardium in the rat myocardial ischemia preconditioning (IPC) model. METHOD: Infarct size was measured by Masson's Trichrome staining, the expression of protein and mRNA of adiponectin at 0, 6, 12 and 24 h after IPC was examined by immunohistochemistry and quantitative real time RT-PCR, plasma levels of adiponectin at above mentioned four time points after IPC were detected by ELISA in IPC and MI rats. RESULT: Infarct size was smaller in IPC than in MI rats (20% ± 2% vs. 31% ± 3%, P < 0.05). The expression of adiponectin mRNA at 6 h and 12 h after IPC was 2.2 and 2.1 times higher than in Sham rats at respective time points (P < 0.05). Immunohistochemistry staining evidenced increased adiponectin expression in the ischemic area and weak expression of adiponectin in non-ischemic area (P < 0.05). Compared to the sham group, the plasma level of adiponectin increased significantly at 0, 6 and 12 h after IPC (0 h: 7.40 ± 0.47 vs. 10.90 ± 1.74; 6 h: 8.18 ± 1.41 vs. 10.98 ± 1.74; 12 h: 6.97 ± 1.02 vs. 9.31 ± 0.96, P < 0.05). CONCLUSION: IPC reduced infarction size, upregulated the myocardial expression of adiponectin at mRNA and protein levels, and increased plasma adiponectin concentration, suggesting that the adiponectin may play a critical role in the protective effect of IPC.

Formation of engineered intersubunit disulfide bond in cytochrome bc1 complex disrupts electron transfer activity in the complex.

Protein domain movement of the Rieske iron-sulfur protein has been speculated to play an essential role in the bifurcated oxidation of ubiquinol catalyzed by the cytochrome bc1 complex. To better understand the electron transfer mechanism of the bifurcated ubiquinol oxidation at Qp site, we fixed the head domain of ISP at the cyt c1 position by creating an intersubunit disulfide bond between two genetically engineered cysteine residues: one at position 141 of ISP and the other at position 180 of the cyt c1 [S141C(ISP)/G180C(cyt c1)]. The formation of a disulfide bond between ISP and cyt c1 in this mutant complex is confirmed by SDS-PAGE and Western blot. In this mutant complex, the disulfide bond formation is concurrent with the loss of the electron transfer activity of the complex. When the disulfide bond is released by treatment with beta-mercaptoethanol, the activity is restored. These results further support the hypothesis that the mobility of the head domain of ISP is functionally important in the cytochrome bc1 complex. Formation of the disulfide bond between ISP and cyt c1 shortens the distance between the [2Fe-2S] cluster and heme c1, hence the rate of intersubunit electron transfer between these two redox prosthetic groups induced by pH change is increased. The intersubunit disulfide bond formation also decreases the rate of stigmatellin induced reduction of ISP in the fully oxidized complex, suggesting that an endogenous electron donor comes from the vicinity of the b position in the cytochrome b.

Domain conformational switch of the iron-sulfur protein in cytochrome bc1 complex is induced by the electron transfer from cytochrome bL to bH.

Intensive biochemical, biophysical and structural studies of the cytochrome (cyt) bc(1) complex in the past have led to the formulation of the "protonmotive Q-cycle" mechanism for electron and proton transfer in this vitally important complex. The key step of this mechanism is the separation of electrons during the oxidation of a substrate quinol at the Q(P) site with both electrons transferred simultaneously to ISP and cyt b(L) when the extrinsic domain of ISP (ISP-ED) is located at the b-position. Pre-steady state fast kinetic analysis of bc(1) demonstrates that the reduced ISP-ED moves to the c(1)-position to reduce cyt c(1) only after the reduced cyt b(L) is oxidized by cyt b(H). However, the question of how the conformational switch of ISP-ED is initiated remains unanswered. The results obtained from analysis of inhibitory efficacy and binding affinity of two types of Q(P) site inhibitors, Pm and Pf, under various redox states of the bc(1) complex, suggest that the electron transfer from heme b(L) to b(H) is the driving force for the releasing of the reduced ISP-ED from the b-position to c(1)-position to reduce cyt c(1).

Novel mitochondrial DNA mutations associated with Chinese familial hypertrophic cardiomyopathy.

1. Hypertrophic cardiomyopathy (HCM) is a genetic disorder that has a complex set of symptoms and potentially devastating consequences. Increasing evidence indicates that mitochondrial DNA (mtDNA) mutations are responsible for the development of HCM, but the mtDNA mutations appear to differ considerably among different populations and regions. 2. In the present study, three families with HCM were found and investigated: one in Shandong province and two in the Chongqing region of China. The entire mtDNA genome from the 18 affected and 66 unaffected family members was sequenced directly and the mtDNA mutations were determined. 3. The frequency of haplogroup M10 was significantly higher in family members with HCM (HCM group) than in unaffected family members (normal group). Three mtDNA mutations were found with a significantly higher frequency in affected individuals than in unaffected family individuals, namely G7697A in the cytochrome c oxidase subunit II gene (P < 0.0001; odds ratio (OR) 227.5; 95% confidence interval (CI) 23.6­2194.8) and T12477C (P = 0.0037; OR 5.6; 95% CI 1.8­17.6) and G13135A in the NADH dehydrogenase 5 gene (P < 0.0001; OR 26.0; 95% CI 6.9­98.3), suggesting that these mutations are probably associated with susceptibility to HCM. In addition, mitochondrial Complex I activity was markedly decreased in the HCM group, suggesting that these mutations most likely affect mitochondrial respiratory function. 4. In conclusion, the results of the present study imply that mtDNA mutations G7697A, T12477C and G13135A are genetic factors that indicate a susceptibility to HCM and that could be used for the large-scale screening of genetic markers as well as the early diagnosis of HCM.

Asymmetric Dimethylarginine Predicts One-year Recurrent Cardiovascular Events: Potential Biomarker of "Toxin Syndrome" in Coronary Heart Disease.

OBJECTIVE: To examine the prognostic value of serum levels of asymmetric dimethylarginine (ADMA) in patients with stable coronary heart disease (CHD) thus explore a potential biomarker of "toxin syndrome" in CHD. METHODS: In this prospective nested case-control study, 36 of 1,503 Chinese patients with stable CHD experienced at least 1 recurrent cardiovascular event (RCE) during 1-year follow-up. Serum levels of ADMA at the start of follow-up were compared between these 36 cases and 36 controls which matched to cases in terms of gender, age, history of hypertension, and myocardial infarction. RESULTS: Based on the crude model, subjects in the 2 highest ADMA quartiles showed significantly higher risk of developing RCE than those in the lowest ADMA quartile [odds ratio (OR) 4.09, 95% confidence interval (CI) 1.01 to 16.58; OR 6.76, 95% CI 1.57 to 29.07]. This association was also observed in the case-mix model (OR 5.51, 95% CI 1.23 to 24.61; OR 7.83, 95% CI 1.68 to 36.41) and multivariable model (OR 6.64, 95% CI 1.40 to 31.49: OR 13.14, 95% CI 2.28 to 75.71) after adjusting for confounders. The multivariable model which combined ADMA and high-sensitivity C-reactive protein (hsCRP) showed better predictive power with areas under the receiver operator characteristic curves (0.779) than the model of either ADMA (0.694) or hsCRP (0.636). CONCLUSION: Serum ADMA level may be a potential biomarker of "toxin syndrome" in CHD which shows favorable prognostic value in predicting 1-year RCE in patients with stable CHD. [The registration number is ChiCTR-PRNRC-07000012].