Induction of tolerogenic dendritic cells by activated TGF-ß/Akt/Smad2 signaling in RIG-I-deficient stemness-high human liver cancer cells.

BACKGROUND: Dendritic cells (DCs) alter their role from being immunostimulatory to immunosuppressive at advanced stages of tumor progression, but the influence of cancer stem cells (CSCs) and their secreted factors on generation and phenotypic change of DCs is unknown. Retinoic acid-inducible gene I (RIG-I) plays a role in regulation of other cellular processes including leukemic stemness besides its antiviral function. METHODS: Short hairpin RNA-mediated gene silencing was employed to generate stable RIG-I-knocked-down human hepatocellular carcinoma (HCC) cell lines. Expression levels of genes and proteins in spheres of those HCC cells were determined by quantitative real-time PCR and Western bot, respectively. Levels of secreted cytokines were measured by ELISA. The surface molecule expression levels of DCs were analyzed using flow cytometry. The ability of DCs to induce proliferation of T cells was assessed by a mixed lymphocyte reaction (MLR) assay. RESULTS: RIG-I-knocked-down HCC cells showed upregulated expression of stem cell marker genes, enhanced secretion of factors suppressing in vitro generation of DCs into the conditioned medium (CM), and induction of a phenotype of tumor-infiltrating DCs (TIDCs) with low levels of DC markers in their tumors in nude mice. Those DCs and TIDCs showed reduced MLR, indicating RIG-I deficiency-induced immunotolerance. The RIG-I-deficient HCC cells secreted more TGF-ß1 than did reference cells. The tumors formed after injection of RIG-I-deficient HCC cells had higher TGF-ß1 contents than did tumors derived from control cells. DC generation and MLR suppressed by the CM of RIG-I-deficient HCC cells were restored by an anti-TGF-ß1 antibody. TGF-ß1-induced phosphorylation of Smad2 and Akt was enhanced in RIG-I-deficient HCC spheres, knockdown of AKT gene expression abolishing the augmentation of TGF-ß1-induced Smad2 phosphorylation. Akt and p-Akt were co-immunoprecipitated with Smad2 in cytoplasmic proteins of RIG-I-deficient spheres but not in those of control spheres, the amounts of co-immunoprecipitated Akt and p-Akt being increased by TGF-ß stimulation. CONCLUSIONS: Our results demonstrate that RIG-I deficiency in HCC cells induced their stemness, enhanced secretion and signaling of TGF-ß1, tolerogenic TIDCs and less generation of DCs, and the results suggest involvement of TGF-ß1 in those RIG-I deficiency-induced tolerogenic changes and involvement of CSCs in DC-mediated immunotolerance.

Restoration of stemness-high tumor cell-mediated suppression of murine dendritic cell activity and inhibition of tumor growth by low molecular weight oyster polysaccharide.

Dendritic cells (DCs) play key regulatory roles in tumor immunity: increased activity of DCs infiltrating tumor tissues leads to enhancement of tumor immunity. Functions of DCs are also modulated by tumor cell-derived factors. Here, we investigated the effects of low molecular weight oyster polysaccharide (LMW-OPS) on differentiation and function of bone marrow-derived DCs (BMDCs) exposed to a conditioned medium (CM) obtained from spheres of stemness-high colorectal cancer cell lines CMT93 and CT26. The CM containing a detectable level of TGF-ß1 was found to down-regulate the surface expression of major histocompatibility complex class II of BMDCs and to inhibit the potency of BMDCs to stimulate T cells. Those suppressions were partly restored and completely restored by addition of anti-TGF-ß1 and LMW-OPS, respectively. Production of IFN-γ during allogeneic T cell responses was inhibited by the CM, whereas production of TGF-ß1 was augmented by the CM. The IFN-γ profile was also reversed by addition of LMW-OPS. Nuclear translocation of ß-catenin, but not that of NF-κB p65, was induced by TGF-ß1. NF-κB p65 nuclear translocation, but not ß-catenin nuclear translocation, was induced by LMW-OPS. Intraperitoneal injection of LMW-OPS significantly suppressed tumor growth in syngeneic tumor models using CMT93 and CT26 sphere cells, whereas it had no inhibitory effect on the proliferation of either cell line. The results demonstrated that LMW-OPS relieved stemness-high tumor cell-mediated suppression of BMDC function and indicated the in vivo anti-tumor activity of LMW-OPS in which re-stimulation of the activity of DCs infiltrating tumor tissues is presumed to be involved.

Augmentation of Cytolytic Activity in Murine Natural Killer Cells and Inhibition of Tumor Growth by the Ethanol Fraction of Oyster Extract.

A reduced number and/or reduced activity of natural killer (NK) cells, which are important for defense against a variety of cancers and viral infections, occur under various stress conditions and in patients with various diseases. In this article, we report that the 30% to 50% ethanol precipitate of oyster extract (EPOE50) dose-dependently enhanced the activity of mouse spleen NK cells in vitro and in vivo. The activity of EPOE50 was eluted with a molecular weight of about 2000 by gel filtration and was inactivated by periodate but not by proteinase K. The activity of highly purified NK cells was also augmented by EPOE50 but not by oligodeoxyribonucleotide 1585, which mimics bacterial DNA. Administration of EPOE50 to mice stimulated splenic NK cell activity without a change in splenic NK cell populations. Although the proliferation of B16 tumor cells in vitro was slightly stimulated by EPOE50, the growth of B16 melanoma in vivo was dose-dependently suppressed by administration of EPOE50. Taken together, our results indicate that EPOE50 augmented NK cell activity and that its administration to mice inhibited tumor growth presumably through the activation of NK cells and also suggest that the active substance is a sugar-containing oligomer or polymer and is not of bacterial origin.

Protection of free radical-induced cytotoxicity by 2-O-α-D-glucopyranosyl-L-ascorbic acid in human dermal fibroblasts.

The stable ascorbic acid (AA) derivative, 2-O-α-D-glucopyranosyl-L-ascorbic acid (AA-2G), exhibits vitamin C activity after enzymatic hydrolysis to AA. The biological activity of AA-2G per se has not been studied in detail, although AA-2G has been noted as a stable source for AA supply. The protective effect of AA-2G against the oxidative cell death of human dermal fibroblasts induced by incubating with 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) for 24 h was investigated in this study. AA-2G showed a significant protective effect against the oxidative stress in a concentration-dependent manner. AA-2G did not exert a protective effect during the initial 12 h of incubation, but had a significant protective effect in the later part of the incubation period. Experiments using a α-glucosidase inhibitor and comparative experiments using a stereoisomer of AA-2G confirmed that AA-2G had a protective effect against AAPH-induced cytotoxicity without being converted to AA. Our results provide an insight into the efficacy of AA-2G as a biologically interesting antioxidant and suggest the practical use of AA-2G even before being converted into AA as a beneficial antioxidant.

Dextran sulfate-induced degradation of spontaneously apoptotic B cells.

Mature resting mouse splenic B cells undergo spontaneous apoptosis in vitro, unless rescued by specific agents including interleukin 4 and protein kinase C activators. Dextran sulfate, a B cell activator, has been reported to have such a protective effect on B cell apoptosis. This study was undertaken to elucidate the mechanism underlying the protective effect of dextran sulfate. The ratio of apoptotic B cells gradually increased to about one third with 24 h of incubation. Dextran sulfate dose-dependently reduced apoptotic cells, but it did not cause concomitant increase in viable cells. Both DNA levels and lactate dehydrogenase activities in the supernatants of dextran sulfate-treated cultures were significantly higher than those in the supernatants of untreated cultures. Concomitantly, DNA levels and lactate dehydrogenase activities in the cell pellets of dextran sulfate-treated cultures were lower than those in the cell pellets of untreated cultures. Addition of dextran sulfate to the culture of B cells 18 h after the start of incubation, when about one fifth of the B cells were dead, significantly reduced apoptotic cells during the next 6-h incubation. This decrease in the number of apoptotic cells was detectable as early as 1 h after addition of dextran sulfate and was prevented by Zn(2+), Co(2+), Ni(2+), the serine protease inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF) or incubation on ice. These results indicated that dextran sulfate treatment did not prevent apoptosis but rather promoted degradation of apoptotic cells and suggest the involvement of DNase and protease in this process.

Induction of neurite outgrowth in PC12 cells by artemisinin through activation of ERK and p38 MAPK signaling pathways.

Growth of neurite processes is a critical step in neuronal development, regeneration, differentiation, and response to injury. The discovery of compounds that can stimulate neurite formation would be important for developing new therapeutics against both neurodegenerative disorders and trauma-induced neuronal injuries. Semisynthetic derivatives of artemisinin, an active compound in Artemisia annua, have been effectively used in malaria treatment, but they have been shown to possess neurotoxic potential. In this study, we found unexpectedly that artemisinin and its derivatives induced neurite outgrowth of PC12 cells. Artemisinins containing an endoperoxide bridge such as artemisinin and dihydroartemisinin induced growth of neurite processes at concentrations that were slightly cytotoxic, artemisinin having the most potent maximal effect among them. Deoxyartemisinin, which lacks the endoperoxide bridge, was ineffective. Artemisinin-treated cells expressed increased levels of the neuronal marker ß(III)-tubulin. Artemisinin upregulated phosphorylation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK), critical signaling molecules in neuronal differentiation. Consistent with activation of the two MAPKs, neurite outgrowth induced by artemisinin was inhibited by the MAPK/ERK kinase inhibitor PD98059 and the p38 MAPK inhibitor SB203580. Artemisinin also induced phosphorylation of cyclic AMP response element-binding protein (CREB) that was almost completely attenuated by PD98059 but not by SB203580. Taken together, our results indicate that artemisinin and its derivatives containing the endoperoxide bridge induced differentiation of PC12 cells toward a neuronal phenotype and suggest that both activation of ERK signaling pathway, which leads to CREB phosphorylation, and activation of p38 MAPK signaling pathway are involved in this process.

Mitogen-activated protein kinases-dependent induction of hepatocyte growth factor production in human dermal fibroblasts by the antibiotic polymyxin B.

Hepatocyte growth factor (HGF) stimulates migration and proliferation of keratinocytes and has been suggested to be involved in wound healing. The cationic antibiotic polymyxin B (PMB) is commonly used as a topical antibiotic for wound care. If PMB possesses an HGF-inducing activity, the antibiotic is potentially beneficial for wound healing in addition to minimizing chances of infection. In this study, we found that PMB markedly induced HGF production from various types of cells including human dermal fibroblasts. Its effect was stronger than the effects of epidermal growth factor and cholera toxin and was comparable to the effect of 8-bromo-cAMP. Among the polymyxin family and polymyxin derivatives, colistin was also effective, whereas colistin methanesulfonate had only a marginal effect and PMB nonapeptide was ineffective. The stimulatory effect of PMB was accompanied by upregulation of HGF gene expression. Increase in phosphorylation of extracellular signal-regulated protein kinase (ERK) and c-Jun N-terminal kinase (JNK) was observed from 0.25 h to 6h after the addition of PMB, while increase in phosphorylation of p38 mitogen-activated protein kinase (MAPK) was detected from 24h to 60 h after PMB addition. The MAPK/ERK kinase inhibitor PD98059, the JNK inhibitor SP600125 and the p38 MAPK inhibitor SB203580 all potently inhibited PMB-induced HGF production. Lastly, proliferation of human dermal fibroblasts was significantly stimulated by PMB. These results indicate that PMB-induced HGF production and proliferation of human dermal fibroblasts and suggest that activation of MAPKs is involved in the induction of HGF production.

Restriction of mast cell proliferation through hyaluronan synthesis by co-cultured fibroblasts.

Appropriate culture models for tissue mast cells are required to determine how they are involved in regulation of local immune responses. We previously established a culture model for cutaneous mast cells, in which bone marrow-derived immature mast cells were co-cultured with Swiss 3T3 fibroblasts in the presence of stem cell factor. In this study, we focused on the roles of hyaluronan, which is produced by the feeder fibroblasts and forms the extracellular matrix during the co-culture period. Hyaluronan synthesis was found to be mediated by hyaluronan synthase 2 (HAS2) expressed in Swiss 3T3 cells. A decreases in the amount of hyaluronan, which was achieved by retroviral expression of short hairpin RNA for Has2 or by addition of hyaluronidase, significantly enhanced the proliferation of the cultured mast cells without any obvious effects on their maturation. Although we previously demonstrated that CD44 is required for proliferation of cutaneous mast cells, the deficiency of hyaluronan did not affect the proliferation of the cultured mast cells that lack CD44. These findings suggest that the extracellular matrix containing hyaluronan may have a potential to restrict proliferation of cutaneous mast cells in a CD44-independent manner.

Promotion of IL-4- and IL-5-dependent differentiation of anti-mu-primed B cells by ascorbic acid 2-glucoside.

The stable ascorbic acid derivative 2-O-alpha-D-glucopyranosyl-L-ascorbic acid (AA-2G) was used to investigate the role of ascorbic acid (AA) in B cell differentiation in vitro. AA-2G is stable in a solution unlike AA but is hydrolyzed by cellular alpha-glucosidase to release AA. Mouse spleen B cells were primed for 2 days with an anti-mu antibody in the presence of interleukin (IL)-4 and IL-5 and then washed and recultured with AA-2G in the presence of IL-4 and IL-5. AA-2G, but not AA, dose-dependently increased IgM production, the greatest enhancement being 150% at concentrations of more than 0.5mM. In the absence of IL-4 and IL-5, primed B cells produced a negligible amount of IgM, and AA-2G had no effect. AA-2G-induced IgM production in the presence of IL-4 and IL-5 was inhibited by the alpha-glucosidase inhibitor castanospermine. Intracellular AA content, depleted during the priming period, increased by adding AA-2G at the start of reculture. Treatment of B cells with AA-2G resulted in an increase in the number of IgM-secreting cells, CD138-positive cells and CD45R/B220-negative cells. The number of viable cells in untreated cultures decreased gradually, but the decrease was significantly attenuated by AA-2G, resulting in about 70% more viable cells in AA-2G-treated cultures. AA-2G caused a slight but reproducible enhancement of DNA synthesis and a slight decrease in the number of cells with a sub-G1 DNA content. These results demonstrated that AA released from AA-2G enhanced cytokine-dependent IgM production in anti-mu-primed B cells and suggest that its effect is caused through promoting the differentiation of B cells to plasma cells and attenuating the gradual decrease in the number of viable cells.

Induction of hepatocyte growth factor production in human dermal fibroblasts and their proliferation by the extract of bitter melon pulp.

Hepatocyte growth factor (HGF) is useful as a potential therapeutic agent for hepatic and renal fibrosis and cardiovascular diseases through inducing proliferation of epithelial and endothelial cells. HGF inducers may also be useful as therapeutic agents for these diseases. However, there have been no reports on induction of HGF production by plant extracts or juices. An extract of bitter melon (Momordica charantia L.) pulp markedly induced HGF production. There was a time lag of 72 h before induction of HGF production after the extract addition. Its stimulatory effect was accompanied by upregulation of HGF gene expression. Increases in mitogen-activated protein kinases (MAPKs) were observed from 72 h after the extract addition. Inhibitors of MAPKs suppressed the extract-induced HGF production. The extract also stimulated cell proliferation. Both activities for induction of HGF production and cell proliferation were eluted together in a single peak with 14,000 Da on gel filtration. The results indicate that bitter melon pulp extract induced HGF production and cell proliferation of human dermal fibroblasts and suggest that activation of MAPKs is involved in the HGF induction. Our findings suggest potential usefulness of the extract for tissue regeneration and provide an insight into the molecular mechanism underlying the wound-healing property of bitter melon.

Induction of hepatocyte growth factor expression by maleic acid in human fibroblasts through MAPK activation.

Carboxylic acids have various biological activities and play critical roles in cellular metabolic pathways such as the tricarboxylic acid (TCA) cycle. It has been shown that some carboxylic acids induce cell proliferation and production of cytokines or growth factors. However, there have been no reports on effects of carboxylic acids on hepatocyte growth factor (HGF) expression. In this study, we found that only maleic acid among various carboxylic acids examined markedly induced HGF production from human dermal fibroblasts. Maleic acid also induced HGF production from human lung fibroblasts and neuroblastoma cells. The stimulatory effect was accompanied by upregulation of HGF gene expression. Increase in phosphorylation of extracellular signal-regulated protein kinase (ERK) and c-Jun N-terminal kinase (JNK) but not in phosphorylation of p38 was observed from 6 h and up to 24 h after maleic acid addition. The ERK kinase inhibitor PD98059 and the JNK inhibitor SP600125 potently inhibited maleic acid-induced HGF production, while the p38 inhibitor SB203580 did not significantly inhibit the production. The protein synthesis inhibitor cycloheximide completely inhibited upregulation of HGF mRNA induced by maleic acid but superinduced HGF mRNA expression upregulated by 12-O-tetradecanoylphorbol 13-acetate (TPA). These results suggest that maleic acid indirectly induced HGF expression from human dermal fibroblasts through activation of ERK and JNK and that de novo protein synthesis is required for maleic acid-induced upregulation of HGF mRNA.

Induction of cytolytic activity and interferon-gamma production in murine natural killer cells by polymyxins B and E.

Natural killer (NK) cells are the primary effector cells of the innate immune system and have well-established roles in tumor rejection and resistance to viruses, bacteria and certain parasites. There is a need for more specific immune modulators of NK cell activity that lack the wide-ranging side effects of NK cell-stimulatory interleukins. The polycationic antibiotic polymyxin B (PMB) has been shown to have a unique ability to enhance activities of some immune cells, independent of its antibiotic properties. Here we report that both PMB and its analog polymyxin E (PME) markedly enhanced the activity of NK cells enriched from the murine spleen. Maximal activation of NK cell activity was obtained after 24 h of incubation with PMB at a dose of 300 mug/ml. PMB nonapeptide, one of the two PMB domains, and PME methanesulfonate, the negatively charged derivative of PME, had little effect on NK cell activity. PMB induced interferon (IFN)-gamma and tumor necrosis factor-alpha production in NK cells. Proliferation of NK cells in vitro was significantly stimulated by being incubated with PMB. Administration of PMB to mice for 7 consecutive days stimulated splenic NK cell activity and increased NK cell populations in the spleen. These results suggest that the polycationic antibiotics PMB and PME may up-regulate innate and adaptive immune responses by induction of NK cell activity and IFN-gamma production.

Inhibition of tumor-stromal interaction through HGF/Met signaling by valproic acid.

Hepatocyte growth factor (HGF), which is produced by surrounding stromal cells, including fibroblasts and endothelial cells, has been shown to be a significant factor responsible for cancer cell invasion mediated by tumor-stromal interactions. We found in this study that the anti-tumor agent valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, strongly inhibited tumor-stromal interaction. VPA inhibited HGF production in fibroblasts induced by epidermal growth factor (EGF), platelet-derived growth factor, basic fibroblast growth factor, phorbol 12-myristate 13-acetate (PMA) and prostaglandin E(2) without any appreciable cytotoxic effect. Other HDAC inhibitors, including butyric acid and trichostatin A (TSA), showed similar inhibitory effects on HGF production stimulated by various inducers. Up-regulations of HGF gene expression induced by PMA and EGF were also suppressed by VPA and TSA. Furthermore, VPA significantly inhibited HGF-induced invasion of HepG2 hepatocellular carcinoma cells. VPA, however, did not affect the increases in phosphorylation of MAPK and Akt in HGF-treated HepG2 cells. These results demonstrated that VPA inhibited two critical processes of tumor-stromal interaction, induction of fibroblastic HGF production and HGF-induced invasion of HepG2 cells, and suggest that those activities serve for other anti-tumor mechanisms of VPA besides causing proliferation arrest, differentiation, and/or apoptosis of tumor cells.

Inhibition of free radical-induced erythrocyte hemolysis by 2-O-substituted ascorbic acid derivatives.

Inhibitory effects of 2-O-substituted ascorbic acid derivatives, ascorbic acid 2-glucoside (AA-2G), ascorbic acid 2-phosphate (AA-2P), and ascorbic acid 2-sulfate (AA-2S), on 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH)-induced oxidative hemolysis of sheep erythrocytes were studied and were compared with those of ascorbic acid (AA) and other antioxidants. The order of the inhibition efficiency was AA-2S> or =Trolox=uric acid> or =AA-2P> or =AA-2G=AA>glutathione. Although the reactivity of the AA derivatives against AAPH-derived peroxyl radical (ROO(*)) was much lower than that of AA, the derivatives exerted equal or more potent protective effects on AAPH-induced hemolysis and membrane protein oxidation. In addition, the AA derivatives were found to react per se with ROO(*), not via AA as an intermediate. These findings suggest that secondary reactions between the AA derivative radical and ROO(*) play a part in hemolysis inhibition. Delayed addition of the AA derivatives after AAPH-induced oxidation of erythrocytes had already proceeded showed weaker inhibition of hemolysis compared to that of AA. These results suggest that the AA derivatives per se act as biologically effective antioxidants under moderate oxidative stress and that AA-2G and AA-2P may be able to act under severe oxidative stress after enzymatic conversion to AA in vivo.

Determination of ascorbic acid and its related compounds in foods and beverages by hydrophilic interaction liquid chromatography.

A new hydrophilic interaction liquid chromatography method for the simultaneous determination of ascorbic acid (AA), erythorbic acid (EA), 2-O-alpha-D-glucopyranosyl-L-ascorbic acid (AA-2G) and 2-O-beta-D-glucopyranosyl-L-ascorbic acid (AA-2betaG) was developed using a diol column with an isocratic solution of acetonitrile-66.7 mM ammonium acetate solution (85:15, v/v) at a detection wavelength of 260 nm. The calibration curves were found to be linear in the range of 1-50 microg/ml for AA and EA and in the range of 2.5-100 microg/ml for AA-2G and AA-2betaG. Detection limits of AA, EA, AA-2G and AA-2betaG were 0.3, 0.3, 0.03 and 0.03 microg/ml, respectively. This method was satisfactorily applied to the determination of AA, EA, AA-2G and AA-2betaG in a fruit, a food and beverages. The results show that the procedure is simple and sensitive and that it can be employed for the simultaneous determination of AA and its related compounds in foods and beverages.

2-O-alpha-D-glucopyranosyl-L-ascorbic acid scavenges 1,1-diphenyl-2-picrylhydrazyl radicals via a covalent adduct formation.

The 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging mechanism of 2-O-alpha-D-glucopyranosyl-L-ascorbic acid (AA-2G) was studied. We found two undefined products, named X and Y, in the reaction mixture of AA-2G and the DPPH radical under acidic conditions by HPLC analysis. The reaction mixture was further subjected to LC-MS analysis. X was found to be a covalent adduct of AA-2G and the DPPH radical. On the other hand, Y could not be identified, probably because it was a mixture. A time-course study of the radical-scavenging reaction revealed that one molecule of AA-2G scavenged one molecule of DPPH radical to generate an AA-2G radical, which readily reacted with another molecule of the DPPH radical to form a covalent adduct (X). Subsequently, this adduct slowly quenched a third molecule of the DPPH radical, resulting in reaction products (Y). Therefore, one molecule of AA-2G has only one oxidizable -OH group, but can scavenge three molecules of the DPPH radical. The radical-scavenging mechanism of AA-2G elucidated in this study should be useful in understanding the biological roles of AA-2G per se in the food and cosmetic fields.

Differentiation of murine B cells induced by chondroitin sulfate B.

A two-step culture system was used to investigate the role of chondroitin sulfate (CS) B, which is mitogenic to B cells, in differentiation of B cells. Mouse spleen B cells were incubated for 3 days with CSB in the presence of interleukin (IL)-4 and IL-5. After washing, the cells were replated at 10(5) viable cells/well and recultured without CSB in the presence of IL-4 and IL-5. CSB dose-dependently increased IgM production, the greatest enhancement being 450%. Dextran sulfate had a similar effect, whereas other glycosaminoglycans, CSA, CSC, heparin and hyaluronic acid, were marginally effective. Treatment of B cells with CSB resulted in increases in the number of IgM-secreting cells and numbers of CD138-positive cells and CD45R/B220-negative cells. CSB-induced IgM production was inhibited by the protein kinase C (PKC) inhibitor GF109203X but not by the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin. These results demonstrated that CSB promoted differentiation of B cells in the presence of IL-4 and IL-5 and suggested that PKC but not PI3K is crucial for CSB-induced IgM production.

An isocratic HPLC method for the simultaneous determination of novel stable lipophilic ascorbic acid derivatives and their metabolites.

2-O-alpha-D-glucopyranosyl-6-O-hexadecanoyl-L-ascorbic acid (6-sPalm-AA-2G), a novel stable lipophilic ascorbic acid derivative, was hydrolyzed to 2-O-alpha-D-glucopyranosyl-L-ascorbic acid (AA-2G), ascorbyl 6-palmitate (6-sPalm-AA) and ascorbic acid (AA) with alpha-glucosidase and lipase. An HPLC method for the simultaneous determination of AA, AA-2G, 6-sPalm-AA and 6-sPalm-AA-2G was developed using a cyanopropyl column with an isocratic solution of methanol-phosphate buffer (pH 2.1) (65:35, v/v) containing 20mg/l of dithiothreitol at a detection wavelength of 240 nm. The calibration curves were found to be linear in the range of 10-200 microM. Linear regression analysis of the data demonstrated the efficacy of the method in terms of precision and accuracy. This method was satisfactorily applied to the determination of 6-sPalm-AA-2G and its three metabolites in a 6-sPalm-AA-2G solution treated with purified enzymes or a small intestine post-mitochondrial supernatant and to the separation of novel stable lipophilic AA derivatives other than 6-sPalm-AA-2G and their metabolites. AA, AA-2G and other well-known stable AA derivatives, ascorbic acid 2-phosphate and ascorbic acid 2-sulfate, were also separated under the same conditions. The results show that the procedure is rapid and simple and that it can be employed for in vitro metabolic analysis of various AA derivatives.

Characterization of the radical-scavenging reaction of 2-O-substituted ascorbic acid derivatives, AA-2G, AA-2P, and AA-2S: a kinetic and stoichiometric study.

The aim of this study was to characterize the antioxidant activity of three ascorbic acid (AA) derivatives O-substituted at the C-2 position of AA: ascorbic acid 2-glucoside (AA-2G), ascorbic acid 2-phosphate (AA-2P), and ascorbic acid 2-sulfate (AA-2S). The radical-scavenging activities of these AA derivatives and some common low molecular-weight antioxidants such as uric acid or glutathione against 1,1-diphenyl-picrylhydrazyl (DPPH) radical, 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation (ABTS+), or galvinoxyl radical were kinetically and stoichiometrically evaluated under pH-controlled conditions. Those AA derivatives slowly and continuously reacted with DPPH radical and ABTS+, but not with galvinoxyl radical. They effectively reacted with DPPH radical under acidic conditions and with ABTS+ under neutral conditions. In contrast, AA immediately quenched all species of radicals tested at all pH values investigated. The reactivity of Trolox, a water-soluble vitamin E analogue, was comparable to that of AA in terms of kinetics and stoichiometrics. Uric acid and glutathione exhibited long-lasting radical-scavenging activity against these radicals under certain pH conditions. The radical-scavenging profiles of AA derivatives were closer to those of uric acid and glutathione rather than to that of AA. The number of radicals scavenged by one molecule of AA derivatives, uric acid, or glutathione was equal to or greater than that by AA or Trolox under the appropriate conditions. These data suggest the potential usage of AA derivatives as radical scavengers.

Development of a rapid semi-quantitative immunochromatographic assay for serum hepatocyte growth factor and its usefulness in acute liver failure.

Measurement of serum human hepatocyte growth factor (HGF) by enzyme-linked immunosorbent assay (ELISA) is useful for the early diagnosis and prediction of prognosis of patients with acute liver failure (ALF). This ELISA methodology, however, is neither rapid nor convenient for use at the bedside. In this study, we have developed a rapid semi-quantitative immunochromatographic (IC) assay and evaluated its usefulness in assessing patients with acute hepatic injury. Only 100mul of serum is required; the assay can be easily completed in 20min. The values obtained using this novel assay correlated well with the values obtained using the standard ELISA protocol. In addition, the values obtained in the IC assay correlated with clinical course; increased serum HGF levels were associated with an increased frequency of ALF and death. These results indicate that this rapid semi-quantitative IC assay for HGF is useful for the early diagnosis of ALF and prediction of clinical outcome in acute hepatic injury.