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Background 4Z,15Z-bilirubin-IXα (BR), an endogenous toxic compound that is sparingly soluble in water, binds human serum albumin (HSA) with high affinity in a flexible manner. Our previous findings suggest that both Lys195 and Lys199 in subdomain IIA are important for the high-affinity binding of BR, and especially Lys199 in stand-alone domain II plays a prominent role in the renal elimination of BR. Our hypothesis is that HSA-domain II with high BR binding would be a useful therapeutic agent to treat hyperbilirubinemia in patients with impaired liver function. Methods Unbound BR concentrations were determined using a modified HRP assay. To evaluate the effect of pan3_3-13 domain II mutant in promoting urinary BR excretion, the serum concentration and urinary excretion amount of BR were determined using bile duct ligation mice. Results After three or six rounds of panning, pan3_3-13 and pan6_4 were found to have a significantly higher affinity for BR than wild-type domain II. Administration of pan3_3-13 significantly reduced serum BR level and increased its urinary excretion in the disease model mice as compared to wild-type domain II treatment. Conclusions These results suggest that pan3_3-13 has great potential as a therapeutic agent that promotes urinary BR excretion in hyperbilirubinemia. General significance This is the first study to be applied to other HSA bound toxic compounds that are responsible for the progression of disease, thereby paving the way for the development of non-invasive and cost effective blood purification treatment methods.

Hydrogen sulfide (H2S) signaling involves polysulfide (RSSnSR') formation on various proteins. However, the current lack of sensitive polysulfide detection assays poses methodological challenges for understanding sulfane sulfur homeostasis and signaling. We developed a novel combined assay by modifying Sulfide Antioxidant Buffer (SAOB) to produce an "Elimination Method of Sulfide from Polysulfide" (EMSP) treatment solution that liberates sulfide, followed with methylene blue (MB) sulfide detection assay. The combined EMSP-MB sulfide detection assay performed on low molecular weight sulfur species showed that sulfide was produced from trisulfide compounds such as glutathione trisulfide and diallyl trisulfide, but not from the thiol compounds such as cysteine, cystine and glutathione. In the case of plasma proteins, this novel combined detection assay revealed that approximately 14.7, 1.7, 3.9, 3.7 sulfide mol/mol released from human serum albumin, α1-anti-trypsin, α1-acid glycoprotein and ovalbumin, respectively, suggesting that serum albumin is a major pool of polysulfide in human blood circulation. Taken together with the results of albumins of different species, the liberated sulfide has a good correlation with cysteine instead of methionine, indicating the site of incorporation of polysulfide is cysteine. With this novel sulfide detention assay, approximately 8,000, 120 and 1100 μM of polysulfide concentrations was quantitated in human healthy plasma, saliva and tear, respectively. Our promising polysulfide specific detection assay can be a very important tool because quantitative determination of polysulfide sheds light on the functional consequence of protein-bound cysteine polysulfide and expands the research area of reactive oxygen to reactive polysulfide species.

Skeletal muscle atrophy, referred to as sarcopenia, is often observed in chronic kidney disease (CKD) patients, especially in patients who are undergoing hemodialysis. The purpose of this study was to determine whether uremic toxins are involved in CKD-related skeletal muscle atrophy. Among six protein-bound uremic toxins, indole containing compounds, indoxyl sulfate (IS) significantly inhibited proliferation and myotube formation in C2C12 myoblast cells. IS increased the factors related to skeletal muscle breakdown, such as reactive oxygen species (ROS) and inflammatory cytokines (TNF-α, IL-6 and TGF-β1) in C2C12 cells. IS also enhanced the production of muscle atrophy-related genes, myostatin and atrogin-1. These effects induced by IS were suppressed in the presence of an antioxidant or inhibitors of the organic anion transporter and aryl hydrocarbon receptor. The administered IS was distributed to skeletal muscle and induced superoxide production in half-nephrectomized (1/2 Nx) mice. The chronic administration of IS significantly reduced the body weights accompanied by skeletal muscle weight loss. Similar to the in vitro data, IS induced the expression of myostatin and atrogin-1 in addition to increasing the production of inflammatory cytokines by enhancing oxidative stress in skeletal muscle. These data suggest that IS has the potential to accelerate skeletal muscle atrophy by inducing oxidative stress-mediated myostatin and atrogin-1 expression.

A ferric citrate formulation for treating hyperphosphatemia is a new therapeutic that not only suppresses the accumulation of phosphorus in patients with chronic kidney disease-mineral bone disorders (CKD-MBD), but also ameliorates anemia caused by iron deficiency. In contrast, it has been demonstrated that intravenous iron injection markedly increases oxidative stress. This study was designed to investigate the effect of a ferric citrate formulation on oxidative stress in CKD-MBD patients receiving hemodialysis therapy. Fifteen CKD-MBD patients undergoing dialysis were enrolled in this study. The patients were orally administered a ferric citrate formulation for 6 months. Their plasma phosphorus concentrations remained unchanged with the switch from other phosphorus adsorbents to the ferric citrate formulation. In addition, the ferric citrate formulation generally allowed for dose reduction of an erythropoiesis stimulating agent with an increased hematopoietic effect. The average values of plasma ferritin level increased after the introduction of a ferric citrate formulation, but did not exceed 100 (ng/mL). Interestingly, oxidative stress markers did not increase significantly, and anti-oxidative capacity was not significantly decreased at 6 months after the drug administration. Similarly, no change was observed in any inflammation markers. The ferric citrate formulation induces negligible oxidative stress in CKD-MBD patients receiving dialysis under the present clinical condition.

3-Carboxy-4-methyl-5-propyl-2-furanpropionate (CMPF), a furan fatty acid uremic toxin (UT) and a substrate for organic ion transporters, contributes to the accumulation of CMPF in renal tubular cells. Although oxidative stress induced by UTs has been proposed as a mechanism of its toxicity in chronic kidney disease, little information is available regarding the redox property of CMPF and its relation to renal cell damage. The findings herein show that CMPF enhances the production of reactive oxygen species (ROS) in HK-2 cells in the presence of angiotensin II (A-II), an inducer of O(2)(·-). When iron is also present, CMPF and A-II induce the Fenton reaction, resulting in a further increase in ROS production. Such CMPF-induced oxidative stress increases TGF-β1 secretion in HK-2 cells, and a positive correlation between CMPF-induced ROS production and the secretion of active TGF-β1 was observed. CMPF caused a reduction in cell viability which was negatively correlated with intracellular ROS production. These negative effects of CMPF in HK-2 cells were completely suppressed by probenecid, an inhibitor of organic anion transport. Interestingly, in vitro ROS assays indicate that CMPF directly interacts with superoxide anion radicals (O(2)(·-)) and peroxy radicals (LOO) to produce CMPF radicals. The subsequent interaction of CMPF radicals with dissolved oxygen leads to the overproduction of O(2)(·-). Based on these findings, we conclude that CMPF, which accumulates in the renal cells, appears to play a prominent role as a pro-oxidant which subsequently leads to renal cellular damage via the overproduction of O(2)(·-).

Human serum albumin (HSA) is the most abundant circulating protein and its S-nitrosated form serves as a reservoir of nitric oxide (NO). Previously, we prepared poly-S-nitrosated HSA (Poly-SNO-HSA) by incubation with Traut's Reagent and isopentyl nitrite and evaluated its potential as a novel anticancer agent through apoptosis involving the caspase-3 pathway. Recently, NO donors such as nitroglycerin were reported to revert the resistance to anticancer agents. Therefore, now we have evaluated the effect of the above type of Poly-SNO-HSA on the resistance to doxorubicin (dx) in human myelogenous leukemic cells (K562 cells). P-gp expression and dx accumulation in K562 and dx-resistant K562 cells (K562/dx cells) were quantified using Western blot and FACS analysis, respectively. Compared with parent K562 cells, higher expression of P-gp and lower accumulation of dx were shown in K562/dx cells. Poly-SNO-HSA caused increased dx accumulation in K562/dx cells by decreasing the expressions of P-gp and HIF-1α. Other experiments with the guanylate cyclase inhibitor ODQ and 8-Br-cGMP revealed that also a cGMP signaling pathway is involved in the Poly-SNO-HSA induced increase in dx accumulation. Furthermore, in vivo studies showed that co-treatment with Poly-SNO-HSA enhanced the anticancer effect of dx in K562/dx cells-bearing mice. Thus, in addition to its proapoptotic effect Poly-SNO-HSA can in an efficient manner revert drug resistance both in vitro and in vivo, and two pathways for this effect have been identified.

Recently, p-cresyl sulfate (PCS) has been identified as a protein-bound uremic toxin. Moreover, the serum-free concentration of PCS, which is associated with its efficacy of hemodialysis, appears to be a good predictor of survival in chronic kidney disease (CKD). We previously found that PCS interacts with indoxyl sulfate (IS), another sulfate-conjugated uremic toxin, during renal excretion via a common transporter. The purpose of this study was to further investigate the interaction between PCS and IS on the binding to human serum albumin (HSA). Here, we used ultrafiltration to show that there is only one high-affinity binding site for PCS, with a binding constant on the order of 10(5) M(-1) (i.e., comparable to that of IS). However, a binding constant of the low-affinity binding site for PCS is 2.5-fold greater than that for IS. Displacement of a fluorescence probe showed that PCS mainly binds to site II, which is the high-affinity site for PCS, on HSA. This finding was further supported by experiments using mutant HSA (R410A/Y411A) that displayed reduced site II ligand binding. A Klotz analysis showed that there could be competitive inhibition between PCS and IS on HSA binding. A similar interaction between PCS and IS on HSA was also observed under the conditions mimicking CKD stage 4 to 5. The present study suggests that competitive interactions between PCS and IS in both HSA binding and the renal excretion process could contribute to fluctuations in their free serum concentrations in patients with CKD.

Summary The importance of cysteine (Cys) and methionine (Met) residues for the antioxidant activity of human serum albumin (HSA) was investigated using recombinant HSA mutants, in which Cys34 and/or the six Met residues had been mutated to Ala. The scavenging activities of the mutants against five reactive oxygen and nitrogen species were evaluated by a chemiluminescence assay, electron paramagnetic resonance spectroscopy, or a HPLC-flow reactor assay. Our results showed that the contributions of Cys34 and the Met residues to the antioxidant activity of HSA were 61% and 29% against O2 � 2, 68% and 61% against H2O2, 38% and 6% against HO � , 36% and 13% against HOCl, and 51% and 1% against � NO, respectively. Thus, the findings propose in a direct way that Cys34 plays a more important role than the Met residues in the antioxidant activity of HSA. 2012 IUBMB IUBMB Life, 64(5): 450–454, 2012

Macromolecules have been developed as carriers of low-molecular-weight drugs in drug delivery systems (DDS) to improve their pharmacokinetic profile or to promote their uptake in tumor tissue via enhanced permeability and retention (EPR) effects. In the present study, recombinant human serum albumin dimer (AL-Dimer), which was designed by linking two human serum albumin (HSA) molecules with the amino acid linker (GGGGS)(2), significantly accumulated in tumor tissue even more than HSA Monomer (AL-Monomer) and appearing to have good retention in circulating blood in murine colon 26 (C26) tumor-bearing mice. Moreover, we developed S-nitrosated AL-Dimer (SNO-AL-Dimer) as a novel DDS compound containing AL-Dimer as a carrier, and nitric oxide (NO) as (i) an anticancer therapeutic drug/cell death inducer and (ii) an enhancer of the EPR effect. We observed that SNO-AL-Dimer treatment induced apoptosis of C26 tumor cells in vitro, depending on the concentration of NO. In in vivo experiments, SNO-AL-Dimer was found to specifically deliver large amounts of cytotoxic NO into tumor tissue but not into normal organs in C26 tumor-bearing mice as compared with control (untreated tumor-bearing mice) and SNO-AL-Monomer-treated mice. Intriguingly, S-nitrosation improved the uptake of AL-Dimer in tumor tissue through augmenting the EPR effect. These data suggest that SNO-AL-Dimer behaves not only as an anticancer therapeutic drug, but also as a potentiator of the EPR effect. Therefore, SNO-AL-Dimer would be a very appealing carrier for utilization of the EPR effect in future development of cancer therapeutics.

4Z,15Z-bilirubin-IXα (4Z,15Z-BR), an endogenous compound that is sparingly soluble in water, binds human serum albumin (HSA) with high affinity in a flexible manner. A phage library displaying recombinant HSA domain II was constructed, after three rounds of panning against immobilized 4Z,15Z-BR, and eight clones with high affinity for the pigment were found to contain conserved basic residues, such as lysine or arginine, at positions 195 and 199. The wild type and two mutants, K195A and K199A, of whole HSA as well as stand-alone domain II were expressed in Pichia pastoris for ligand-binding studies. The binding of 4Z,15Z-BR to the K195A and K199A mutants was decreased in both whole HSA and the domain II proteins. The P-helicity conformer (P-form) of 4Z,15Z-BR was found to preferentially bind to the wild types and the K195A mutants, whereas the M-form bound to the K199A mutants. Photoconversion experiments showed that the P-form of 4Z,15Z-BR was transformed into highly water-soluble isomers at a much faster rate than the M-form. In addition, the M-form of 4Z,15Z-BR showed higher affinity for domain I than for domain II. The present findings suggest that, whereas both Lys195 and Lys199 in subdomain IIA are important for the high-affinity binding of 4Z,15Z-BR, Lys199 plays a more prominent role in the elimination of 4Z,15Z-BR.

S -Nitrosated human serum albumin (SNO-HSA) is a large molecular weight nitric oxide carrier in human plasma, and because of its many beneficial effects in different tests, it is currently under investigation as a cytoprotective agent. However, making SNO-HSA preparations is a complicated and time-consuming process. We found that binding of caprylic acid (CA) and N-acetyl- l -tryptophan (N-AcTrp) to defatted mercaptalbumin increased S- nitrosation by S -nitrosoglutathione (GS-NO) by making Cys-34 of HSA more accessible and by protecting it against oxidation, respectively. Fortunately, HSA solutions for clinical use contain high concentrations of CA and N-AcTrp as stabilizers. By making use of that fact it was possible to work-out a fast and simple procedure for producing SNO-HSA: incubation of a commercial HSA formulation with GS-NO for only 1 min results in S- nitrosation of HSA. The biological usefulness of such a preparation was tested in a rat ischemia–reperfusion liver injury model. Although our procedure for making SNO-HSA is fast and straightforward, the cytoprotective effect of the preparation was similar to, or better than, that of a preparation made in a more traditional way. The clinical development of SNO-HSA as a strong cytoprotective agent is under way using this method in collaboration with clinicians and industrial developers.

Human serum albumin (HSA), a non-glycosylated protein, is widely employed as carrier for drug delivery systems. A series of recombinant, mannosylated-HSA mutants (Man-rHSAs: D63N, A320T and D494N) and their triple mutant (TM-rHSA: D63N/A320T/D494N) were prepared, that can be selectively delivered to the liver via mannose receptor (MR) on the liver non-parenchymal cells. A pharmacokinetic analysis of (111)In-Man-rHSAs in mice showed that they were rapidly cleared from the blood circulation, and were largely taken up by the liver rapidly in the order: TM-rHSA>D494N>>A320T=D63N, consistent with their degree of mannosylation. In vivo competition experiments with an excess amount of chemically modified Man-BSA or mannan suggested that the hepatic uptake of TM-rHSA was selectively mediated by MR on Kupffer cells. Lastly, a TM-rHSA-NO conjugate, S-nitrosylated TM-rHSA, effectively delivered NO to the liver and then exhibited a significant inhibitory effect against hepatic ischemia/reperfusion injury model rats, accompanied by the induction of heme oxygenase-1.

Recently, nitric oxide has been investigated as a potential anti-cancer therapy because of its cytotoxic activity. Previously, we found that S-nitrosylated human serum albumin (SNO-HSA) induced apoptosis in C26 cells, demonstrating for the first time that SNO-HSA has potential as an anti-cancer drug. In the present study, the anti-tumor activity of SNO-HSA in another tumor type of cancer cell was investigated using murine tumor LY-80 cells. Mitochondrial depolarization, activation of caspase-3 and DNA fragmentation were induced in LY-80 cells by SNO-HSA treatment in a dose-dependent manner. Inhibition of caspase activity resulted in complete inhibition of DNA fragmentation induced by SNO-HSA. The cytotoxic effects of SNO-HSA on LY-80 were concentration-dependent. Tumor growth in LY-80-tumor-bearing rats was significantly inhibited by administration of SNO-HSA compared with saline- and HSA-treatment. These results suggest that SNO-HSA has potential as a chemopreventive and/or chemotherapeutic agent because it induces apoptosis in tumor cells.

Recently,substantial epidemiological research has clarified that folic acid decreases the incidence of fetal dysraphia such as spina bifida and anencephaly.Consequently,the Ministry of Health,Labour and Welfare (MHLW) now recommends that women of child-bearing age take folic acid proactively.However,previous consumer surveys on folic acid performed by many organizations have shown that pregnant women have little knowledge of the usefulness of folic acid,and few women of child-bearing age take folic acid on initiative.In our research,we carried out a questionnaire survey on awareness of folic acid among females aged 10-39 years and provided information on appropriate folic acid intake.The information was provided by community pharmacists and we verified the effectiveness of doing this.In the initial questionnaire,many subjects selected“don’t know”for all items concerning folic acid surveyed.In particular,hardly any knew that folic acid reduces the risk of congenital abnormality occurrence,and that the MHLW recommends that child-bearing women take folic acid.One month after providing people with information on folic acid,the results of a survey showed that their understanding of it had increased significantly for all items but in a similar survey conducted after six months,indicated that their understanding had decreased significantly for several items,as compared to results for one month after receiving information.These results showed that a health promotion intervention by community pharmacists raised consumer awareness and strongly suggested that this is an effective approach in primary care practice.However,the temporary nature of this effect suggested that there is a need for repeated provision of information.

Summary: Nitric oxide (NO) is a ubiquitous molecule involved in multiple cellular functions. Inappropriate production of NO may lead to disease states. To date, pharmacologically active compounds that release NO within the body, such as organic nitrates, have been used as therapeutic agents, but their efficacy is significantly limited by rapid NO release, toxicity and induction of tolerance. Therefore, novel NO donors with better pharmacological and phamacokinetic properties are highly desirable. The S-nitrosothiol fraction in plasma is largely composed of S-nitrosylated human serum albumin (SNO-HSA) and that is why we are testing whether this albumin form can be used as a NO traffic protein. We have found that oleate and other endogenous ligands increase SNO-HSA formation in vitro. The cytoprotective effect of SNO-HSA in a ischemia/reperfusion model and its antiapoptotic effect on HepG2 cells treated with anti-Fas antibody were pronounced and could be enhanced by binding of oleate. The enhancement of S-transnitrosation to the HepG2 cells could be completely blocked by filipin III, a caveolae inhibitor. These findings indicate that a clinical application of SNO-HSA is expected as potent NO supplementary therapy and that fatty acids may serve as novel types of mediators for S-transnitrosation.

Recently, human serum albumin (HSA) has emerged as a versatile carrier for therapeutic agents against diabetes, cancer, and infectious diseases. Market-approved products include fatty acid derivatives of human insulin for diabetes and the paclitaxel-HSA nanoparticle for various cancers such as metastatic breast cancer and advanced pancreatic cancer. In this review, we focus on the next-generation approach including HSA-binding bioactive gas such as nitric oxide (NO) for treating ischemic/reperfusion injury, cancer, and bacterial infection. To date, pharmacologically active compounds that release NO within the body, such as organic nitrates, have been used as therapeutic agents, but their efficacy is significantly limited by unwanted side effects. Therefore, novel NO donors with better pharmacological and pharmacokinetic properties are highly desirable. The S-nitrosothiol fraction in plasma is largely composed of endogenous S-nitrosated HSA (SNO-HSA), which is why we are investigating whether this albumin form can be therapeutically useful. Recently, we have developed SNO-HSA analogues such as SNO-HSA with many conjugated SNO groups (poly-SNO-HSA) prepared using chemical modification. Unexpectedly, we found striking inverse effects between poly-SNO-HSA and SNO-HSA. Despite the fact that SNO-HSA inhibits apoptosis, poly-SNO-HSA possesses very strong pro-apoptotic effects against tumor cells. Furthermore, poly-SNO-HSA can reduce or even completely eliminate the multidrug resistance often developed by cancer cells. In this review, we put forward the possibility that poly-SNO-HSA can be used as a safe, effective multifunctional antitumor agent.

Nitric oxide (NO) is a ubiquitous messenger molecule that is involved in multiple cellular functions. In particular, NO has multiple important actions that contribute to the maintenance of vascular homeostasis. Thus, an inappropriate production of NO leads to a disease state. To date, pharmacologically active compounds that release NO within the body, such as organic nitrates, have been used as therapeutic agents, but their efficacy is significantly limited by undesirable side effects. Therefore, novel NO donors with better pharmacological and pharmacokinetic properties would be highly desirable. The S-nitrosothiol fraction in plasma is largely composed of endogenous S-nitrosated human serum albumin (Mono-SNO-HSA), and that is why we are testing whether this albumin form has the potential to be therapeutically useful. Recently, we developed SNO-HSA analogs such as SNO-HSA with many conjugated SNO groups (Poly-SNO-HSA) which were prepared using chemical modification. Unexpectedly, we found striking inverse effects between Poly-SNO-HSA and Mono-SNO-HSA. Despite the fact that Mono-SNO-HSA inhibits apoptosis, Poly-SNO-HSA exerts very strong proapoptotic effects against tumor cells. Furthermore, Poly-SNO-HSA can reduce or perhaps completely eliminate the multidrug resistance often developed by cancer cells. In addition, we recently developed an S-nitrosated HSA dimer (SNO-HSA dimer) as a novel enhanced permeability and retention (EPR) effect enhancer. The SNO-HSA dimer increases the tumor accumulation of macromolecular antitumor drugs by a factor 3-4 and thereby their antitumor effects. In this review, we first summarize the effective factors on the S-nitrosation and S-denitrosation of HSA both in vitro and in biological systems. Finally, we propose the possibility that Poly-SNO-HSA and SNO-HSA dimer can be used as a safe and effective multifunctional antitumor agent and as a nano-EPR enhancer.

Many proteins have been identified as targets for S-nitrosylation, including structural and signaling proteins, a nd ion channels. S-nitrosylation plays an important role in regulating their activity and function. We used human serum albumin (HS A), a major endogenous NO traffic protein, and studied the effect of mediators on S-nitrosylation processes which control NO bioactivity. By using NOC-7, S-nitrosoglutathione, and activated RAW264.7 cells as NO-donors we found that high-affinity binding of endogenous ligands (Cu(2+), bilirubin and fatty acid) can affect these processes. It is likely that the same effects take place in many clinical situations characterized by increased fatty acid concentrations in plasma such as type II diabetes and the metabolic syndrome. Thus, endogenous ligands, changing their plasma concentrations, could be a novel type of mediator of S-nitrosylation not only in the case of HSA but also for other target proteins.

BACKGROUND: Natural mutations of R218 in human serum albumin (HSA) result in an increased affinity for L-thyroxine and lead to the autosomal dominant condition of familial dysalbuminemic hyperthyroxinemia.METHODS: Binding was studied by equilibrium dialysis and computer modelling.RESULTS: 10 of 32 other isoforms tested had modified high-affinity hormone binding. L-thyroxine has been reported to bind to four sites (Tr) in HSA; Tr1 and Tr4 are placed in the N-terminal and C-terminal part of the protein, respectively. Site-directed mutagenesis gave new information about all the sites.CONCLUSIONS: It is widely assumed that Tr1 is the primary hormone site, and that this site, on a modified form, is responsible for the above syndrome, but the binding experiments with the genetic variants and displacement studies with marker ligands indicated that the primary site is Tr4. This new assignment of the high-affinity site was strongly supported by results of MM-PBSA analyses and by molecular docking performed on relaxed protein structure. However, dockings also revealed that mutating R218 for a smaller amino acid increases the affinity of Tr1 to such an extent that it can become the high-affinity site.GENERAL SIGNIFICANCE: Placing the high-affinity binding site (Tr4) and the one which can result in familial dysalbuminemic hyperthyroxinemia (Tr1) in two very different parts of HSA is not trivial, because in this way persons with and without the syndrome can have different types of interactions, and thereby complications, when given albumin-bound drugs. The molecular information is also useful when designing drugs based on L-thyroxine analogues.

Peroxynitrite, the reaction product of superoxide [Formula: see text] and nitric oxide (NO), nitrates tyrosine residues, unsaturated fatty acids, cyclic guanosine monophosphate and other phenolics. We report herein that indoxyl sulfate (IS) is also nitrated by peroxynitrite in vitro and forms 2-nitro-IS, as determined from spectral characteristics and (1)H-NMR. IS is one of the very important uremic toxins that accelerate the progression of chronic kidney disease via various mechanisms. However, cell viability experiments with human proximal tubular cells show that the cytotoxicity of 2-nitro-IS is several-fold higher than that of IS. The explanation for this finding seems to be that 2-nitro-IS induces a much more pronounced generation of intracellular reactive oxygen species (ROS) than IS. Results with inhibitors revealed that an organic anion transporter, several intracellular enzymes and nonprotein-bound iron ions are reasons for this finding. Most importantly, however, as detected by immunofluorescence and Western blotting, 2-nitro-IS induces the expression of heme oxygenase-1 and thereby the formation of ROS; most probably through the Fenton reaction. The final result of the increased amounts of ROS is death of the kidney cells. Thus, nitration of uremic toxins by peroxynitrite may help us to understand the initiation and progress of chronic kidney diseases.

The S-nitrosylated forms of certain proteins such as albumin have been thought to be circulating endogenous reservoirs of nitric oxide (NO) and may have potential as NO donors in therapeutic applications. In this study, we investigated the characteristics of R410C, a genetic variant of human serum albumin with two free thiols at positions 34 (Cys-34) and 410 (Cys-410), as a NO carrier via S-nitroso formation. A biotin switch assay revealed that Cys-410 was more rapidly and efficiently nitrosylated than was Cys-34. Nitrosylation of Cys-410 introduced only small conformational changes in the protein, which were detected by far-UV circular dichroism but not by near-UV circular dichroism. In addition, both native R410C and S-nitrosylated R410C did not induce molecular heterogeneity through oligomerization. S-Nitrosylated R410C exhibited strong antibacterial activity against Salmonella typhimurium in vitro and suppressed apoptosis of U937 human promonocytic cells induced by Fas ligand. In a rat ischemia-reperfusion liver injury model, S-nitrosylated R410C treatment significantly reduced liver damage, as indicated by markedly decreased release of liver enzymes (aspartate aminotransferase and alanine aminotransferase). Pharmacokinetic analyses indicated retention of the S-nitroso moiety of S-nitrosylated R410C in circulation after i.v. injection, with an approximate half-life of 20.4 min in the mouse. These data suggest that R410C can be a useful NO carrier and can be regarded as a new class of S-nitrosylated proteins possessing antibacterial and cytoprotective properties with a circulation time sufficient for in vivo biological activity.

Since reactive oxygen species (ROS) derived from Kupffer cells (KC), especially CD68(+) KC, play a key role in the induction of hepatic oxidative stress and injuries, we developed a polythiolated- and mannosylated human serum albumin (SH-Man-HSA), which functions as a novel nanoantioxidant for delivering thiol to CD68(+) KC. In vitro electron paramagnetic resonance coupled with pharmacokinetics and immunohistochemical studies showed that SH-Man-HSA possessed powerful radical-scavenging activity and rapidly and selectively delivered thiols to the liver via mannose receptor (CD206) on CD68(+) cells. SH-Man-HSA significantly improved the survival rate of concanavalin-A (Con-A)-treated mice. Moreover, SH-Man-HSA exhibited excellent hepatoprotective functions, not by decreasing tumor necrosis factor or interferon-γ production that is closely associated with Con-A-induced hepatitis, but by suppressing ROS production. Interestingly, the protective effect of SH-Man-HSA was superior to N-acetyl cysteine (NAC). This could be attributed to the difference in the inhibition of hepatic oxidative stress between the two antioxidants depending on their potential for thiol delivery to the liver. Similar results were also observed for acetaminophen (APAP)-induced hepatopathy models. Flow cytometric data further confirmed that an increase in F4/80(+)/ROS(+) cells was dramatically decreased by SH-Man-HSA. The administration of SH-Man-HSA at 4 hours following a Con-A or APAP injection also exhibited a profound hepatoprotective action against these hepatitis models, whereas this was not observed for NAC. It can be concluded therefore that SH-Man-HSA has great potential for use in a rescue therapy for hepatopathy as a nanoantioxidant because of its ability to efficiently and rapidly deliver thiols to CD68(+)/CD206(+) KC.

Bone marrow is a key element in the diagnosis of disorders of erythropoiesis, including anemia, and a potential target in their treatment. However, because efficient delivery of diagnostic and therapeutic agents to bone marrow is difficult, such delivery is achieved by administering drugs in large quantities that often have adverse effects. Here, we achieved selective delivery of recombinant human erythropoietin (rHuEPO) to bone marrow, via its encapsulation in liposomes with l-glutamic acid, N-(3-carboxy-1-oxopropyl)-, 1,5-dihexadecyl ester (SA) (liposome-EPO). The result, in a rabbit model of renal anemia, was a beneficial effect on hematopoiesis, better than with rHuEPO alone. Also, we determined that liposome-EPO delivery to bone marrow depended on specific uptake by bone marrow macrophages because of the presence of SA. These results indicate both that liposome-EPO is a new, promising erythropoietin-stimulating agent and that liposomes with SA have potential for diagnostic and therapeutic applications in diseases originating from bone marrow.

The major cause of death in patients with chronic kidney disease (CKD) is cardiovascular disease. Here, p-Cresyl sulfate (PCS), a uremic toxin, is considered to be a risk factor for cardiovascular disease in CKD. However, our understanding of the vascular toxicity induced by PCS and its mechanism is incomplete. The purpose of this study was to determine whether PCS enhances the production of reactive oxygen species (ROS) in vascular endothelial and smooth muscle cells, resulting in cytotoxicity. PCS exhibited pro-oxidant properties in human umbilical vein endothelial cells (HUVEC) and aortic smooth muscle cells (HASMC) by enhancing NADPH oxidase expression. PCS also up-regulates the mRNA levels and the protein secretion of monocyte chemotactic protein-1 (MCP-1) in HUVEC. In HASMC, PCS increased the mRNA levels of alkaline phosphatase (ALP), osteopontin (OPN), core-binding factor alpha 1, and ALP activity. The knockdown of Nox4, a subunit of NADPH oxidase, suppressed the cell toxicity induced by PCS. The vascular damage induced by PCS was largely suppressed in the presence of probenecid, an inhibitor of organic anion transporters (OAT). In PCS-overloaded 5/6-nephrectomized rats, plasma MCP-1 levels, OPN expression, and ALP activity of the aortic arch were increased, accompanied by the induction of Nox4 expression. Collectively, the vascular toxicity of PCS can be attributed to its intracellular accumulation via OAT, which results in an enhanced NADPH oxidase expression and increased ROS production. In conclusion, we found for the first time that PCS could play an important role in the development of cardiovascular disease by inducing vascular toxicity in the CKD condition.

In this report, the authors described the unusual case of a patient in whom the plasma phenytoin concentration was unexpectedly not detected on a particle-enhanced turbidimetric inhibition immunoassay (PETINIA) technique, a typical immunoassay for phenytoin. The plasma concentration was measured using PETINIA and high-performance liquid chromatography in a 69-year-old male patient treated with fosphenytoin intravenously at the standard dose for 7 days. Although the plasma concentration of phenytoin was below the limit of detection (

Macromolecules have been developed as carriers of low-molecular-weight drugs in drug delivery systems (DDS) to improve their pharmacokinetic profile or to promote their uptake in tumor tissue via enhanced permeability and retention (EPR) effects. We have previously demonstrated that poly-nitric oxide (NO) conjugated human serum albumin (Poly-SNO-HSA) has the potential to be a DDS carrier capable of accumulating NO in tumors. However, the stability of Poly-SNO-HSA in the circulation has to be improved, and its optimal molecular size for using the EPR effects has to be evaluated. In the present study, we performed two tuning methods for refining Poly-SNO-HSA, namely, pegylation and dimerization. We observed that pegylation enhanced the stability of Poly-SNO-HSA both in vitro and in vivo, and that dimerization of Poly-SNO-HSA enhanced the antitumor activity via more efficient delivery of NO in Colon 26 tumor-bearing mice. Intriguingly, dimerization resulted in a 10 times higher antitumor activity. These data suggest that pegylation and dimerization of Poly-SNO-HSA are very important tuners to optimize NO stability and accumulation, and thereby effect, in tumors. Thus, polyethylene glycol-Poly-SNO-HSA dimer seems to be a very appealing and safe NO carrier and thereby a strong candidate as an antitumor drug in future development of cancer therapeutics.

Recent clinical studies have shown that increased serum levels of p-cresyl sulfate (PCS), a uremic toxin, are associated with the progression of chronic kidney disease (CKD) and cardiovascular outcomes. Using rat renal cortical slices, we previously reported that the rat organic anion transporter (OAT) could play a key role in the renal tubular secretion of PCS. However, no information is currently available regarding the transport of PCS via human OAT (hOAT) isoforms, hOAT1 and hOAT3.Uptake experiments of PCS were performed using HEK293 cells, which stably express hOAT1 or hOAT3.PCS was taken up by hOAT1/HEK293 and hOAT3/HEK293 cells in a time- and concentration-dependent manner. The apparent K m for the hOAT1-mediated transport of PCS was 128 μM, whereas in hOAT3/HEK293, saturation was not observed at the highest tested PCS concentration of 5 mM. Probenecid, an OAT inhibitor, inhibited PCS transport by hOAT1 and hOAT3. The uptake of p-aminohippurate by hOAT1 and estron-3-sulfate by hOAT3 was decreased with increasing PCS concentration. The apparent 50 % inhibitory concentrations for PCS were 690 and 485 μM for hOAT1 and hOAT3, respectively.PCS is a substrate for hOAT1 and hOAT3, and hOAT1 and hOAT3 appear to play a physiological role as a high-capacity PCS transporter. Since hOATs are expressed not only in the kidneys, but also in blood vessels and osteoblasts, etc., these findings are of great significance in terms of elucidating the renal clearance, tissue disposition of PCS and the mechanism of its toxicity in CKD.

Idiopathic pulmonary fibrosis (IPF) is thought to involve inflammatory cells and reactive oxygen species (ROS), such as superoxide anion radical (O2(·-)). There is currently no effective treatment of IPF. We previously developed a human serum albumin (HSA)-thioredoxin 1 (Trx) fusion protein (HSA-Trx) designed to overcome the unfavorable pharmacokinetic and short pharmacological properties of Trx, an antioxidative and anti-inflammatory protein. In this study, we examined the therapeutic effect of HSA-Trx on an IPF animal model of bleomycin (BLM)-induced pulmonary fibrosis. A pharmacokinetic study of HSA-Trx or Trx in BLM mice showed that the plasma retention and lung distribution of Trxc was markedly improved by fusion with HSA. A weekly intravenous administration of HSA-Trx, but not Trx, ameliorated BLM-induced fibrosis, as evidenced by a histopathological analysis and pulmonary hydroxyproline levels. HSA-Trx suppressed active-transforming growth factor (TGF)-β levels in the lung and inhibited the increase of inflammatory cells in bronchoalveolar lavage fluid, pulmonary inflammatory cytokines, and oxidative stress markers. An in vitro EPR experiment using phosphate-buffered saline-stimulated neutrophils confirmed the O2(·-) scavenging ability of HSA-Trx. Furthermore, post-treatment of HSA-Trx had a suppressive effect against BLM-induced fibrosis. These results suggest that HSA-Trx has potential as a novel therapeutic agent for IPF, because of its long-acting antioxidative and anti-inflammatory modulation effects.

Contrast-induced nephropathy (CIN), caused by a combination of the direct tubular toxicity of contrast media, a reduction in medullary blood flow, and the generation of reactive oxygen species, is a serious clinical problem. A need exists for effective strategies for its prevention. Thioredoxin-1 (Trx) is a low-molecular-weight endogenous redox-active protein with a short half-life in the blood due to renal excretion. We produced a long-acting form of Trx as a recombinant human albumin–Trx fusion protein (HSA–Trx) and examined its effectiveness in preventing renal injury in a rat model of ioversol-induced CIN. Compared with saline, a mixture of HSA and Trx, or Trx alone, intravenous HSA–Trx pretreatment significantly attenuated elevations in serum creatinine, blood urea nitrogen, and urinary N -acetyl-β-D-glucosaminidase along with the decrease in creatinine clearance. HSA–Trx also caused a substantial reduction in the histological features of renal tubular injuries and in the number of apoptosis-positive tubular cells. Changes in the markers 8-hydroxy deoxyguanosine and malondialdehyde indicated that HSA–Trx significantly suppressed renal oxidative stress. In HK-2 cells, HSA–Trx decreased the level of reactive oxygen species induced by hydrogen peroxide, and subsequently improved cell viability. Thus, our results suggest that due to its long-acting properties, HSA–Trx has the potential to effectively prevent CIN.

Nitric oxide (NO) is a ubiquitous molecule involved in multiple cellular functions. Inappropriate production of NO may lead to disease states. To date, pharmacologically active compounds that release NO within the body, such as organic nitrates, have been used as therapeutic agents, but their efficacy is significantly limited by unwanted side effects. Therefore, novel NO donors with better pharmacological and pharmacokinetic properties are highly desirable. TheS-nitrosothiol fraction in plasma is largely composed of endogenousS-nitrosated human serum albumin (Mono-SNO-HSA), and that is why we are testing whether this albumin form can be therapeutically useful. Recently, we developed SNO-HSA analogs such as SNO-HSA with many conjugated SNO groups (Poly-SNO-HSA) which were prepared using chemical modification. Unexpectedly, we found striking inverse effects between Poly-SNO-HSA and Mono-SNO-HSA. Despite the fact that Mono-SNO-HSA inhibits apoptosis, Poly-SNO-HSA possesses very strong proapoptotic effects against tumor cells. Furthermore, Poly-SNO-HSA can reduce or perhaps completely eliminate the multidrug resistance often developed by cancer cells. In this review, we forward the possibility that Poly-SNO-HSA can be used as a safe and effective multifunctional antitumor agent.

S-Nitrosated human serum albumin (SNO-HSA) is useful in preventing liver ischemia/reperfusion injury, and SNO-HSA should thus be able to prevent cell injury during liver transplantation. However, the potential protective effect of SNO-HSA on a combination of cold and warm ischemia, which is obligatory when performing liver transplantation, has not been examined. Therefore, we evaluated the protective effect of SNO-HSA added to University of Wisconsin (UW) solution during cold or/and warm ischemia in situ and in vitro. First, we observed that apoptotic and necrotic cell death were increased during cold and warm ischemia, respectively. SNO-HSA, which possesses anti-apoptosis activity at low NO concentrations, can inhibit cold ischemia injury both in situ and in vitro. In contrast, SNO-HSA had no significant effect on warm liver ischemia injury which, however, can be reduced by UW solution. We also demonstrated that the cellular uptake of NO from SNO-HSA can occur during cold ischemia resulting in induction of heme oxygenase-1 within 3h of cold ischemia. Our results indicate that treatment with SNO-HSA or UW solution alone is not sufficient to inhibit liver injury during a period of both cold and warm ischemia. However, a combination of SNO-HSA and UW solution can be used to prevent the two types of ischemia. SNO-HSA-added UW solution could be very useful in transplantation, because the previously imposed constraints on preservation time can be removed. This is a great advantage in a situation as the present one with increased utilization of scarce donor organs for more recipients.

Treating infections with exogenous NO, which shows broad-spectrum antimicrobial activity, appears to be effective. Similar to NO biosynthesis, biosynthesis of α-1-acid glycoprotein variant A (AGPa), with a reduced cysteine (Cys149), increases markedly during inflammation and infection. We hypothesized that AGPa is an S-nitrosation target in acute-phase proteins. This study aimed to determine whether S-nitrosated AGPa (SNO-AGPa) may be the first compound of this novel antibacterial class against multidrug-resistant bacteria. AGPa was incubated with RAW264.7 cells activated by lipopolysaccharide and interferon-γ. The antimicrobial effects of SNO-AGPa were determined by measuring the turbidity of the bacterial suspensions in vitro and survival in a murine sepsis model in vivo, respectively. Results indicated that endogenous NO generated by activated RAW264.7 cells caused S-nitrosation of AGPa at Cys149. SNO-AGPa strongly inhibited growth of gram-positive, gram-negative, and multidrug-resistant bacteria and was an extremely potent bacteriostatic compound (IC(50): 10(-9) to 10(-6) M). The antibacterial mechanism of SNO-AGPa involves S-transnitrosation from SNO-AGPa to bacterial cells. Treatment with SNO-AGPa, but not with AGPa, markedly reduced bacterial counts in blood and liver in a mouse sepsis model. The sialyl residues of AGPa seem to suppress the antibacterial activity, since SNO-asialo AGPa was more potent than SNO-AGPa.

8-Nitroguanosine 3′,5′-cyclic monophosphate (8-nitro-cGMP) is a nitric oxide metabolite and an important second messenger. 8-Nitro-cGMP reacts with sulfhydryl groups forming a novel posttranslational modification, namely, S -guanylation. In this work, we found, by using a quantitative competition enzyme-linked immunosorbent assay procedure, that S -guanylated human serum albumin ( S -cGMP-HSA) is a component of normal plasma, and that hemodialysis patients decrease its concentration, on an average, from 68 to 34nM. End-stage renal disease is often accompanied by septicemia, and we found that S -cGMP-HSA possesses an in vitro antibacterial effect with half maximal inhibitory concentration of approximately 2μM against Escherichia coli American Type Culture Collection. Our findings indicate that S -cGMP-HSA can be regarded as an endogenous antibacterial agent in healthy conditions and as a useful new class of antibacterial agents with a circulation time sufficient for in vivo biological activity. The clinical development of S -cGMP-HSA as a safe and strong antibacterial agent arisen from endogenous posttranslational modification would be expected. © 2012 Wiley Periodicals, Inc. and the American Pharmacists Association.

4Z,15Z-bilirubin-IXα (4Z,15Z-BR), an endogenous compound that is sparingly soluble in water, binds human serum albumin (HSA) with high affinity in a flexible manner. A phage library displaying recombinant HSA domain II was constructed, after three rounds of panning against immobilized 4Z,15Z-BR, and eight clones with high affinity for the pigment were found to contain conserved basic residues, such as lysine or arginine, at positions 195 and 199. The wild type and two mutants, K195A and K199A, of whole HSA as well as stand-alone domain II were expressed in Pichia pastoris for ligand-binding studies. The binding of 4Z,15Z-BR to the K195A and K199A mutants was decreased in both whole HSA and the domain II proteins. The P-helicity conformer (P-form) of 4Z,15Z-BR was found to preferentially bind to the wild types and the K195A mutants, whereas the M-form bound to the K199A mutants. Photoconversion experiments showed that the P-form of 4Z,15Z-BR was transformed into highly water-soluble isomers at a much faster rate than the M-form. In addition, the M-form of 4Z,15Z-BR showed higher affinity for domain I than for domain II. The present findings suggest that, whereas both Lys195 and Lys199 in subdomain IIA are important for the high-affinity binding of 4Z,15Z-BR, Lys199 plays a more prominent role in the elimination of 4Z,15Z-BR.

Thioredoxin (Trx) is a redox-active protein with anti-inflammatory effects but with a short half life of 1 h. Genetic fusion of Trx to human serum albumin (HSA) extended its half life without causing significant loss of its biological activities. HSA-Trx caused a decrease in the number of cells in brochoalveolar lavage fluid, the wet/dry ratio and the inflammation at the respiratory tract of the ovalbumin (OVA) induced lung injury model mouse. Three intraperitoneal doses of Trx alone produced the same extent of suppression of those three detrimental effects of OVA as one intravenous dose of HSA-Trx. Inhibition experiments confirmed that reactive oxygen species (ROS) and reactive nitrogen species (RNS) involved in the progression of the injury. HSA-Trx inhibited the production of ROS as confirmed in the EPR experiment, but lung tissue staining suggested that induced nitrogen oxide synthase (iNOS) was not suppressed by the fusion protein. Instead, the production of nitrotyrosine, 8-nitro-cGMP, and 8-hydroxy-2'-deoxyguanosine downstream to the iNOS has been inhibited. This suggested that HSA-Trx produced lung protection effect via different mechanisms from Trx alone. HSA-Trx retains the biological properties of Trx thus has great potential in treating oxidative stress related diseases.

Endogenous S-nitrosated human serum albumin (E-Mono-SNO-HSA) is a large molecular weight nitric oxide (NO) carrier in human plasma, which has shown many beneficial effects in different animal models. To construct more efficient SNO-HSA preparations, SNO-HSA with many conjugated SNO groups has been prepared using chemical modification (CM-Poly-SNO-HSA). We have compared the properties of such a preparation to those of E-Mono-SNO-HSA. Cellular uptake of NO from E-Mono-SNO-HSA partly takes place via low molecular weight thiol, and it results in cytoprotective effects by induction of heme oxygenase-1. By contrast, transfer of NO from CM-Poly-SNO-HSA into the cells is faster and more pronounced. The influx mainly takes place by cell-surface protein disulfide isomerase. The considerable NO inflow results in apoptotic cell death by ROS induction and caspase-3 activation. Thus, increasing the number of SNO groups on HSA does not simply intensify the cellular responses to the product but can also result in very different effects.

A nitrated guanine nucleotide, 8-nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP), is formed via nitric oxide (NO) and causes protein S-guanylation. However, intracellular 8-nitro-cGMP levels and mechanisms of formation of 8-nitro-cGMP and S-guanylation are yet to be identified. In this study, we precisely quantified NO-dependent formation of 8-nitro-cGMP in C6 glioma cells via liquid chromatography-tandem mass spectrometry. Treatment of cells with S-nitroso-N-acetylpenicillamine led to a rapid, transient increase in cGMP, after which 8-nitro-cGMP increased linearly up to a peak value comparable with that of cGMP at 24 h and declined thereafter. Markedly high levels (40 microm) of 8-nitro-cGMP were also evident in C6 cells that had been stimulated to express inducible NO synthase with excessive NO production. The amount of 8-nitro-cGMP generated was comparable with or much higher than that of cGMP, whose production profile slightly preceded 8-nitro-cGMP formation in the activated inducible NO synthase-expressing cells. These unexpectedly large amounts of 8-nitro-cGMP suggest that GTP (a substrate of cGMP biosynthesis), rather than cGMP per se, may undergo guanine nitration. Also, 8-nitro-cGMP caused S-guanylation of KEAP1 in cells, which led to Nrf2 activation and subsequent induction of antioxidant enzymes, including heme oxygenase-1; thus, 8-nitro-cGMP protected cells against cytotoxic effects of hydrogen peroxide. Proteomic analysis for endogenously modified KEAP1 with matrix-assisted laser desorption/ionization time-of-flight-tandem mass spectrometry revealed that 8-nitro-cGMP S-guanylated the Cys(434) of KEAP1. The present report is therefore the first substantial corroboration of the biological significance of cellular 8-nitro-cGMP formation and potential roles of 8-nitro-cGMP in the Nrf2-dependent antioxidant response.

The effect of the uremic solute indoxyl sulfate (IS) on scavenging superoxide anion radicals (O 2 ) generated from both the xanthine/xanthine oxidase (X/XO) system and activated neutrophils was investigated by electron paramagnetic resonance spectroscopy, combined with 2-ethoxycarbonyl2-methyl-3,4-dihydro-2H-pyrrole-1-oxide (EMPO). The findings show that the presence of normalphysiological serum concentrations of IS (0.1–10 lM) resulted in decreased formation of EMPOsuperoxide adduct without affecting XO activity. Furthermore, IS showed scavenging activity against cell-derived O 2 generated from activated neutrophils. In addition, IS also eliminated hydroxyl radicals. These findings suggest that IS acts as a novel endogenous antioxidant under normal-physiological conditions. 2010 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

Thioredoxin-1 (Trx) is a redox-active protein with anti-inflammatory effects. The effect of albumin fusion on the pharmacokinetic and pharmacodynamic properties of Trx was evaluated in this study. The findings indicate that the properties of human serum albumin and the fusion protein are comparable. The fusion protein showed similar plasma concentration and organ distribution profiles as human serum albumin. The fusion protein accumulated in lungs, reaching levels higher than Trx. In an insulin reducing assay, the activity of the fusion protein was 60% of the activity of Trx. However, survival rate of endotoxic shock mice induced by the administration of a lipopolysaccharide and D-galactosamine for fusion protein was double that of Trx. The findings reported herein indicate that the fusion protein is likely to have great clinical applications in areas such as the treatment of reperfusion injuries.

Binding of oleate to S-nitrosylated human serum albumin (SNO-HSA) enhances its cytoprotective effect on liver cells in a rat ischemia/reperfusion model. It enhances the antiapoptotic effect of SNO-HSA on HepG2 cells exposed to anti-Fas antibody. To identify some of the reasons for the increased cytoprotective effects, additional experiments were performed with glutathione and HepG2 cells. As indicated by 5,5′-dithiobis-2-nitrobenzoic acid binding, the addition of oleate increased the accessibility of the single thiol group of albumin. Binding of increasing amounts of oleate resulted in increasing and more rapid S-transnitrosation of glutathione. Likewise, binding of oleate, or of a mixture of endogenous fatty acids, improved S-denitrosation of SNO-HSA by HepG2 cells. Oleate also enhanced S-transnitrosation by HepG2 cells, as detected by intracellular fluorescence of diaminofluorescein-FM. All of the S-transnitrosation caused by oleate binding was blocked by filipin III. Oleate also increased, in a dose-dependent manner, the binding of SNO-HSA labeled with fluorescein isothiocyanate to the surface of the hepatocytes. A model in two parts was worked out for S-transnitrosation, which does not involve low molecular weight thiols. Fatty acid binding facilitates S-denitrosation of SNO-HSA, increases its binding to HepG2 cells and greatly increases S-transnitrosation by hepatocytes in a way that is sensitive to filipin III. A small nitric oxide transfer takes place in a slow system, which is unaffected by fatty acid binding to SNO-HSA and not influenced by filipin III. Thus, fatty acids could be a novel type of mediator for S-transnitrosation.