Hyperhomocysteinemia is an independent risk factor for cardiovascular disease, complications of pregnancy, cognitive impairment, and osteoporosis. That elevated homocysteine leads to vascular dysfunction may be the linking factor between these apparently unrelated pathologies. Although a growing body of evidence suggests that homocysteine plays a causal role in atherogenesis, specific mechanisms to explain the underlying pathogenesis have remained elusive. This review focuses on chemistry unique to the homocysteine molecule to explain its inherent cytotoxicity. Thus, the high pKa of the sulfhydryl group (pKa, 10.0) of homocysteine underlies its ability to form stable disulfide bonds with protein cysteine residues, and in the process, alters or impairs the function of the protein. Studies in this laboratory have identified albumin, fibronectin, transthyretin, and metallothionein as targets for homocysteinylation. In the case of albumin, the mechanism of targeting has been elucidated. Homocysteinylation of the cysteine residues of fibronectin impairs its ability to bind to fibrin. Homocysteinylation of the cysteine residues of metallothionein disrupts zinc binding by the protein and abrogates inherent superoxide dismutase activity. Thus, S-homocysteinylation of protein cysteine residues may explain mechanistically the cytotoxicity of elevated L-homocysteine.

Ethanol-induced liver injury is characterized by increased formation of reactive oxygen species (ROS) and inflammatory cytokines, resulting in the development of hepatic steatosis, injury, and cell death by necrosis and apoptosis. Thioredoxin (Trx), a potent antioxidant and antiinflammatory molecule with antiapoptotic properties, protects animals from a number of inflammatory diseases. However, the effects of ethanol on Trx or its role in ethanol-induced liver injury are not known. Female C57BL/6 mice were allowedad libitumaccess to a Lieber-deCarli ethanol diet with 5.4% of calories as ethanol for 2 days to acclimate them to the diet, followed by 2 days with 32.4% of calories as ethanol or pair-fed control diet. Hepatic Trx-1 was decreased by ethanol feeding; daily supplementation with recombinant human Trx (rhTrx) prevented this ethanol-induced decrease. Therefore, we tested the hypothesis that administration of rhTrx during ethanol exposure would attenuate ethanol-induced oxidative stress, inflammatory cytokine production, and apoptosis. Mice were treated with a daily intraperitoneal injection of either 5 g/kg of rhTrx or phosphate-buffered saline (PBS).Conclusion:Ethanol feeding increased accumulation of hepatic 4-hydroxynonenal protein adducts, expression of hepatic tumor necrosis factor α, and resulted in hepatic steatosis and increased plasma aspartate aminotransferase and alanine aminotransferase. In ethanol-fed mice, treatment with rhTrx reduced 4-hydroxynonenal adduct accumulation, inflammatory cytokine expression, decreased hepatic triglyceride, and improved liver enzyme profiles. Ethanol feeding also increased transferase-mediated dUTP-biotin nick-end labeling-positive cells, caspase-3 activity, and cytokeratin-18 staining in the liver. rhTrx treatment prevented these increases. In summary, rhTrx attenuated ethanol-induced increases in markers of oxidative stress, inflammatory cytokine expression, and apoptosis. (Hepatology 2009.)

Objective— More than 70% of circulating homocysteine is disulfide-bonded to protein, but little is known about the specific proteins that bind homocysteine and their function as a consequence of homocysteine binding. Methods and Results— When human plasma was incubated with [ ³⁵ S] l -homocysteine, most of the homocysteine bound to albumin. However, additional homocysteine-binding proteins were detected, and 1 of them comigrated with fibronectin. Treatment with 2-mercaptoethanol removed the bound homocysteine, demonstrating the involvement of disulfide bonding. In contrast, [ ³⁵ S] l -cysteine did not bind to fibronectin. Purified fibronectin bound ≈5 homocysteine molecules per fibronectin dimer. SDS-PAGE of a limited trypsin digestion of homocysteinylated fibronectin showed that several tryptic fragments contained [ ³⁵ S]homocysteine. Sequence analysis demonstrated that the fragments containing bound homocysteine had localized mainly to the C-terminal region, within and adjacent to the fibrin-binding domain. Homocysteinylation of fibronectin significantly inhibited its capacity to bind fibrin by 62% ( P <0.005). In contrast, neither the binding of fibronectin to gelatin nor its capacity to serve as an attachment factor for aortic smooth muscle cells was affected. Conclusions— These results suggest that homocysteine may alter normal thrombosis and delay or interfere with wound healing by impairing the interaction of fibronectin with fibrin.

Objective— L-homocysteine and/or L-homocystine interact in vivo with albumin and other extracellular proteins by forming mixed-disulfide conjugates. Because of its extremely rich cysteine content, we hypothesized that metallothionein, a ubiquitous intracellular zinc-chaperone and superoxide anion radical scavenger, reacts with L-homocysteine and that homocysteinylated-metallothionein suffers loss of function. Methods and Results— ³⁵ S -homocysteinylated-metallothionein was resolved in lysates of cultured human aortic endothelial cells in the absence and presence of reduced glutathione by SDS-PAGE and identified by Western blotting and phosphorimaging. Using zinc-Sepharose chromatography, L-homocysteine was shown to impair the zinc-binding capacity of metallothionein even in the presence of reduced glutathione. L-Homocysteine induced a dose-dependent increase in intracellular free zinc in zinquin-loaded human aortic endothelial cells within 30 minutes, followed by the appearance of early growth response protein-1 within 60 minutes. In addition, intracellular reactive oxygen species dramatically increased 6 hours after L-homocysteine treatment. In vitro studies demonstrated that L-homocysteine is a potent inhibitor of the superoxide anion radical scavenging ability of metallothionein. Conclusion— These studies provide the first evidence that L-homocysteine targets intracellular metallothionein by forming a mixed-disulfide conjugate and that loss of function occurs after homocysteinylation. The data support a novel mechanism for disruption of zinc and redox homeostasis.

Accurate assessment of cobalamin profiles in human serum, cells, and tissues may have clinical diagnostic value. However, non-alkyl forms of cobalamin undergo beta-axial ligand exchange reactions during extraction, which leads to inaccurate profiles having little or no diagnostic value. Experiments were designed to: 1) assess beta-axial ligand exchange chemistry during the extraction and isolation of cobalamins from cultured bovine aortic endothelial cells, human foreskin fibroblasts, and human hepatoma HepG2 cells, and 2) to establish extraction conditions that would provide a more accurate assessment of endogenous forms containing both exchangeable and non-exchangeable beta-axial ligands. The cobalamin profile of cells grown in the presence of [ 57Co]-cyanocobalamin as a source of vitamin B12 shows that the following derivatives are present: [ 57Co]-aquacobalamin, [ 57Co]-glutathionylcobalamin, [ 57Co]-sulfitocobalamin, [ 57Co]-cyanocobalamin, [ 57Co]-adenosylcobalamin, [ 57Co]-methylcobalamin, as well as other yet unidentified corrinoids. When the extraction is performed in the presence of excess cold aquacobalaminacting as a scavenger cobalamin (i.e. "cold trapping"), the recovery of both [ 57Co]-glutathionylcobalamin and [ 57Co]-sulfitocobalamin decreases to low but consistent levels. In contrasts, the [ 57Co]-nitrocobalamin observed in the extracts prepared without excess aquacobalamin is undetected in extracts prepared with cold trapping. This demonstrates that beta-ligand exchange occur with non-covalently bound beta-ligands. The exception to this observation is cyanocobalamin with a non-exchangeable CN- group. It is now possible to obtain accurate profiles of cellular cobalamin.