High density lipoprotein (HDL) cholesterol levels are associated with decreased risk of cardiovascular disease, but not all HDL are functionally equivalent. A primary determinant of HDL functional status is the conformational adaptability of its main protein component, apoA-I, an exchangeable apolipoprotein. Chemical modification of apoA-I, as may occur under conditions of inflammation or diabetes, can severely impair HDL function and is associated with the presence of cardiovascular disease. Chemical modification of apoA-I also impairs its ability to exchange on and off HDL, a critical process in reverse cholesterol transport. In this study, we developed a method using electron paramagnetic resonance spectroscopy (EPR) to quantify HDL-apoA-I exchange. Using this approach, we measured the degree of HDL-apoA-I exchange for HDL isolated from rabbits fed a high fat, high cholesterol diet, as well as human subjects with acute coronary syndrome and metabolic syndrome. We observed that HDL-apoA-I exchange was markedly reduced when atherosclerosis was present, or when the subject carries at least one risk factor of cardiovascular disease. These results show that HDL-apoA-I exchange is a clinically relevant measure of HDL function pertinent to cardiovascular disease.

Optical difference spectroscopy was used to identify and quantify human adrenal microsomal and mitochondrial cytochrome P450 enzyme interactions with the histamine H₃ receptor antagonists thioperamide, clobenpropit and ciproxifan. Addition of these structurally diverse imidazole H₃ receptor antagonists to cytochrome-P450-containing human adrenal microsomal and mitochondrial preparations resulted in concentration-dependent type II optical difference spectra. Respective spectral dissociation constants (K_S) for the drug interactions with human adrenal microsomal and mitochondrial cytochrome P450 were 1.5 and 1.6 µmol/l for thioperamide, 3.1 and 0.28 µmol/l for clobenpropit and 0.10 and 0.11 µmol/l for ciproxifan. The three compounds demonstrated a similar activity profile in cytochrome-P450-containing bovine adrenal microsomal and mitochondrial preparations. Findings indicate direct coordination of these imidazole-containing H₃ receptor antagonists with the heme moiety of human adrenal cytochrome P450 isozymes.

An environmental, estrogen-like substance, bisphenol A (BPA), is the monomer for the production of polycarbonate plastics used in baby bottles, dental sealants, and as a major component of epoxy resin for the lining of food cans. The oxidation of BPA leads to the reactive electrophilic BPA-o-3,4-quinone (BPA-Q), which can damage DNA and may be implicated in cancer initiation. BPA-Q reacts in vitro with 2'-deoxyguanosine 5'-phosphate (dGMP) and 2'-deoxyadenosine 5'-phosphate (dAMP) but not with 2'-deoxycytidine-5'-phosphate and 2'-deoxythymidine 5'-phosphate. In aqueous acetic acid, BPA-Q also reacts with 2'-deoxyguanosine (dG) and 2'-deoxyadenosine (dA) but not with 2'-deoxycytidine and 2'-deoxythymidine. The reactions are accompanied by loss of the modified base (depurination). We determined the structures of the modified bases by primarily tandem mass spectrometry. In mixtures of deoxynuclesides and deoxynucletides treated with BPA-Q, reactions occur more readily with dGMP/dG followed by dAMP/dA. With calf thymus DNA, significant apurinic sites must be produced because we detected the BPA-Q-guanosine adduct in the incubation mixture. We also found that BPA-Q reacts readily with glutathione (GSH) under acidic or neutral conditions, and we characterized the BPA-Q-GSH conjugate with tandem mass spectrometry (MS/MS). The results are consistent with a mechanism of carcinogenesis whereby BPA-Q, formed in vivo and not adequately detoxified by reactions with GSH, reacts with DNA, causing depurination. The adducts reported will also be appropriate references for identification of BPA-Q adducts in environmental and biological systems.

BackgroundChronic glucocorticoid excess has been linked to increased atherosclerosis and general cardiovascular risk in humans. The enzyme 11β-hydroxysteroid dehydrogenase type 1 (11βHSD1) increases active glucocorticoid levels within tissues by catalyzing the conversion of cortisone to cortisol. Pharmacological inhibition of 11βHSD1 has been shown to reduce atherosclerosis in murine models. However, the cellular and molecular details for this effect have not been elucidated.Methodology/Principal FindingsTo examine the role of 11βHSD1 in atherogenesis, 11βHSD1 knockout mice were created on the pro-atherogenic apoE−/− background. Following 14 weeks of Western diet, aortic cholesterol levels were reduced 50% in 11βHSD1−/−/apoE−/− mice vs. 11βHSD1+/+/apoE−/− mice without changes in plasma cholesterol. Aortic 7-ketocholesterol content was reduced 40% in 11βHSD1−/−/apoE−/− mice vs. control. In the aortic root, plaque size, necrotic core area and macrophage content were reduced ∼30% in 11βHSD1−/−/apoE−/− mice. Bone marrow transplantation from 11βHSD1−/−/apoE−/− mice into apoE−/− recipients reduced plaque area 39–46% in the thoracic aorta. In vivo foam cell formation was evaluated in thioglycollate-elicited peritoneal macrophages from 11βHSD1+/+/apoE−/− and 11βHSD1−/−/apoE−/− mice fed a Western diet for ∼5 weeks. Foam cell cholesterol levels were reduced 48% in 11βHSD1−/−/apoE−/− mice vs. control. Microarray profiling of peritoneal macrophages revealed differential expression of genes involved in inflammation, stress response and energy metabolism. Several toll-like receptors (TLRs) were downregulated in 11βHSD1−/−/apoE−/− mice including TLR 1, 3 and 4. Cytokine release from 11βHSD1−/−/apoE−/−-derived peritoneal foam cells was attenuated following challenge with oxidized LDL.ConclusionsThese findings suggest that 11βHSD1 inhibition may have the potential to limit plaque development at the vessel wall and regulate foam cell formation independent of changes in plasma lipids. The diminished cytokine response to oxidized LDL stimulation is consistent with the reduction in TLR expression and suggests involvement of 11βHSD1 in modulating binding of pro-atherogenic TLR ligands.

The objective of the present project was to investigate the efficacy and mechanism of acute (10-minute) adenosine treatment for augmentation of ischemic tolerance in muscle flaps in pigs. Varying doses of adenosine were infused into 28 latissimus dorsi muscle flaps through the axillary artery (0, 0.5, or 2.0 mg per flap) and 22 gracilis muscle flaps through the medial circumflex femoral artery (0, 10, or 20 mg per flap) over 10 minutes. Ten minutes after adenosine infusion, these muscle flaps were subjected to 4 hours of sustained warm global ischemia. In addition, one group of latissimus dorsi muscle flaps (n = 6) received a 10-minute intraarterial adenosine infusion (0.5 mg) at the beginning of reperfusion. Muscle biopsies (n = 4 or 5) for adenosine triphosphate (ATP) analysis were obtained before and after adenosine infusion and at the end of 4 hours of ischemia. The extent of muscle infarction was assessed at 48 hours of reperfusion by the tetrazolium dye staining technique. Muscle blood flow in latissimus dorsi muscle flaps was measured at the end of adenosine infusion (0 or 0.5 mg per flap, n = 8) by the radioactive microsphere (15-microns) technique. It was observed that adenosine, at all doses tested, significantly (p < 0.05) reduced the extent of muscle infarction in latissimus dorsi muscle flaps (control, 40.3 +/- 2.2 percent; 0.5 mg, 20.6 +/- 1.6 percent; 2.0 mg, 18.2 +/- 1 percent) and gracilis muscle flaps (control, 31.0 +/- 1.5 percent; 10 mg, 14.3 +/- 3 percent; 20 mg, 11.6 +/- 1.2 percent). Preischemic adenosine treatment (0.5 mg per flap) was associated with maintenance of a significantly (p < 0.05) higher muscle content of ATP in latissimus dorsi muscle flaps at the end of 4 hours of ischemia compared with saline-treated ischemic controls. Postischemic adenosine treatment did not protect latissimus dorsi muscle flaps against infarction. Furthermore, adenosine treatment did not have any significant effect on mean systemic arterial blood pressure or muscle blood flow in latissimus dorsi muscle flaps. It is concluded that acute (10-minute) preischemic adenosine treatment is effective in augmentation of ischemic tolerance in muscle flaps and that this protective effect of adenosine may be, at least in part, the result of slowing muscle ATP depletion during sustained ischemia. The possible mechanisms of this adenosine-induced energysparing effect are discussed.