Journal of Clinical Biochemistry and Nutrition, 2021
Increased 5-hydroxytryptamine may be associated with the development and progression of inflammat... more Increased 5-hydroxytryptamine may be associated with the development and progression of inflammatory bowel disease. In this study, we examined the suppressive effect of flavonoids on the increased intra- and extracellular 5-hydroxytryptamine levels in rat mast RBL-2H3 cells, known to produce 5-hydroxytryptamine by the phorbol 12-myristate 13-acetate stimulation. Among the flavonoids examined, luteolin and quercetin significantly reduced the cellular 5-hydroxytryptamine concentration. Gene and protein expression analyses revealed that luteolin significantly suppressed cellular tryptophan hydroxylase 1 expression induced by phorbol 12-myristate 13-acetate stimulation. Mitogen-activated protein kinase/extracellular signal-regulated kinase signaling was also suppressed by luteolin, suggesting that this pathway is one of targets of 5-hydroxytryptamine modulation by luteolin. An in vivo experimental colitis model was prepared by administering 2.5% dextran sodium sulfate in drinking water ...
Quercetin is a major flavonoid, present as its glycosidic forms in plant foods. In this study, qu... more Quercetin is a major flavonoid, present as its glycosidic forms in plant foods. In this study, quercetin-3-glucoside (Q3G) was administered intraduodenally to thoracic lymph-cannulated rats, and its lymphatic transport was investigated. The resulting lymphatic and plasma metabolites were identified with LC-MS/MS and compared with those after administration of quercetin aglycone. The total concentration of quercetin metabolites in the lymph was about four times lower than that in the plasma, and quercetin and its methylated form isorhamnetin were detected as their glucuronides, sulfates and diglucuronides both in the lymph and the plasma after Q3G and quercetin administrations. The lymph levels of the glucuronides after Q3G administration were lower than those after quercetin administration, whereas those in the plasma showed the opposite pattern. Both the lymph and plasma levels of the sulfates after Q3G administration were lower than those after quercetin administration. Some of th...
Manuka honey, which shows strong nonperoxide-dependent antibacterial activity, contains unique co... more Manuka honey, which shows strong nonperoxide-dependent antibacterial activity, contains unique components, such as methyl syringate 4-O-β-D-gentiobioside (leptosperin) and its aglycone, methyl syringate (MSYR). To determine the potential for biological activity evoked by the ingestion of leptosperin and MSYR, we investigated the absorption and metabolism of these components in manuka honey. The incubation of MSYR with liver microsomes or S9 fractions in vitro resulted in the formation of MSYR-glucuronide (MSYR-GA), MSYR-sulfate (MSYR-S), and syringic acid as metabolites. Then, manuka honey (15 g) was fed to healthy human volunteers. MSYR-GA, MSYR-S, and MSYR were detected in both plasma and urine. Within plasma, their levels were highest within 0.5 h to 1 h post-ingestion, and most metabolites disappeared within 3 h. In conjunction with the disappearances, a significant amount of metabolites along with trace leptosperin was excreted in urine within 4 h. To elucidate the detailed metabolisms of leptosperin and MSYR, each compound was separately administered to mice. In each case, MSYR-GA, MSYR-S, and MSYR were detected in both plasma and urine. This study shows the major molecular pathway for leptosperin and MSYR metabolism and could facilitate an understanding of biological functions of manuka honey post ingestion.
Serotonin (5-hydroxytryptamine) is a putative substrate for myeloperoxidase, which may convert it... more Serotonin (5-hydroxytryptamine) is a putative substrate for myeloperoxidase, which may convert it into the reactive quinone tryptamine-4,5-dione (TD). In this study, we found that the viability of human SH-SY5Y neuroblastoma cells treated with 25 μM TD was increased to approximately 117%. On the other hand, the cell viability was significantly decreased by exposure to TD (150-200 μM), with an increase in intracellular reactive oxygen species (ROS). Interestingly, pre-treatment of SH-SY5Y cells with 100 μM TD prevented cell death and suppressed intracellular ROS generation evoked by the addition of hydrogen peroxide (H2O2). Expression of the phase-II antioxidant enzyme NAD(P)H: quinone oxidoreductase 1 and haem oxygenase 1 were upregulated by TD at a concentration of 50-100 μM. Nuclear factor erythroid 2-related factor 2 (Nrf2), the regulator of these enzyme, was translocated from the cytosol to the nucleus by 100 μM TD. In summary, moderate concentrations of TD may increase the self...
Journal of clinical biochemistry and nutrition, 2016
Myeloperoxidase is an inflammatory enzyme that generates reactive hypochlorous acid in the presen... more Myeloperoxidase is an inflammatory enzyme that generates reactive hypochlorous acid in the presence of hydrogen peroxide and chloride ion. However, this enzyme also uses bromide ion or thiocyanate as a substrate to form hypobromous or hypothiocyanous acid, respectively. These species play important roles in host defense against the invasion of microorganisms. In contrast, these enzyme products modify biomolecules in hosts during excess inflammation, indicating that the action of myeloperoxidase is both beneficial and harmful. Myeloperoxidase uses other endogenous compounds, such as serotonin, urate, and l-tyrosine, as substrates. This broad-range specificity may have some biological implications. Target molecules of this enzyme and its products vary, including low-molecular weight thiols, proteins, nucleic acids, and lipids. The modified products represent biomarkers of myeloperoxidase action. Moderate inhibition of this enzyme might be critical for the prevention/modulation of exce...
Bioscience, biotechnology, and biochemistry, Jan 7, 2016
Lipid peroxidation products react with cellular molecules, such as DNA bases, to form covalent ad... more Lipid peroxidation products react with cellular molecules, such as DNA bases, to form covalent adducts, which are associated with aging and disease processes. Since lipid peroxidation is a complex process and occurs in multiple stages, there might be yet unknown reaction pathways. Here, we analyzed comprehensively 2'-deoxyguanosine (dG) adducts with oxidized arachidonic acid using liquid chromatography-tandem mass spectrometry and found the formation of 7-(2-oxo-hexyl)-etheno-dG as one of the major unidentified adducts. The formation of this adduct was reproduced in the reaction of dG with 2-octenal and predominantly with 4-oxo-2-octenal (OOE). We also found that other 2-alkenals (with five or more carbons) generate corresponding 4-oxo-2-alkenal-type adducts. Importantly, it was found that transition metals enhanced the oxidation of C4-position of 2-octenal, leading to the formation of OOE-dG adduct. These findings demonstrated a new pathway for the formation of 4-oxo-2-alkenals...
Elimination of celiac-toxic prolamin peptides and proteins is essential for Triticeae products to... more Elimination of celiac-toxic prolamin peptides and proteins is essential for Triticeae products to be gluten-free. Instead of enzymatic hydrolysis, in this study we investigated metal-catalyzed oxidation of two model peptides, QQPFP, and PQPQLPY, together with a hordein isolate from barley (Hordeum vulgare L.). We established a multiple reaction monitoring (MRM) LC-MS method to detect and quantify proline oxidation fragments. In addition to fragmentation, aggregation and side chain modifications were identified, including free thiol loss, carbonyl formation, and dityrosine formation. The immunoreactivity of the oxidized hordein isolate was considerably decreased in all metal-catalyzed oxidation systems. Cleavage of peptides or protein fragments at the numerous proline residues partially accounts for the decrease. Metal-catalyzed oxidation can thus be used in the modification and elimination of celiac-toxic peptides and proteins.
Archives of biochemistry and biophysics, Jan 9, 2015
At the sites of inflammation, hypohalous acids, such as hypochlorous acid and hypobromous acid (H... more At the sites of inflammation, hypohalous acids, such as hypochlorous acid and hypobromous acid (HOBr), are produced by myeloperoxidase. These hypohalous acids rapidly react with the primary amino groups to produce haloamines, which are relatively stable and can diffuse long distances and cross the plasma membrane. In this study, we examined the effects of taurine, the most abundant free amino acid in the leukocyte cytosol, on the hypohalous acid-dependent formation of 8-chloro-2'-deoxyguanosine (8-CldG) and 8-bromo-2'-deoxyguanosine (8-BrdG). The reaction of taurine with HOBr yielded taurine bromamine, which is the most stable among other bromamines of amino acids. Taurine also enhanced the bromination of only dG among the four 2'-deoxynucleosides, whereas it inhibited the 8-CldG formation. The specificity of taurine for the enhanced formation of halogenated dG is completely different from that of nicotine, an enhancer of chlorination. The amount of dibrominated taurine ...
Bioscience, biotechnology, and biochemistry, Jan 4, 2015
Myeloperoxidase (MPO)-generated halogenating molecules, such as hypochlorous acid and hypobromous... more Myeloperoxidase (MPO)-generated halogenating molecules, such as hypochlorous acid and hypobromous acid (HOBr), in inflammatory regions are postulated to contribute to disease progression. In this study, we showed that ergothioneine (EGT), derived from an edible mushroom, inhibited MPO activity as well as the formation of 8-bromo-2'-deoxyguanosine in vitro. The HOBr scavenging effect of EGT is higher than those of ascorbic acid and glutathione. We initially observed that the administration of Coprinus comatus, an edible mushroom containing a high amount of EGT, inhibited the UV-B-induced inflammatory responses and DNA halogenation, suggesting that EGT is a promising anti-inflammatory agent from mushrooms.
Inflammatory bowel diseases, including Crohn’s disease and ulcerative colitis, are chronic inflam... more Inflammatory bowel diseases, including Crohn’s disease and ulcerative colitis, are chronic inflammatory disorders associated with oxidative stress. The intestines produce 5-hydroxytryptamine that may negatively affect disease state under inflammatory conditions when overproduced. 5-Hydroxytryptamine is a substrate for myeloperoxidase and is converted into reactive tryptamine-4,5-dione. Here, an experimental colitis model was established through oral administration of 5% dextran sulfate sodium to ICR mice for 7 days. Furthermore, the formation of tryptamine-4,5-dione in the colorectal mucosa/submucosa and colorectal tissue was analyzed by chemical and immunochemical methodologies. First, free tryptamine-4,5-dione in the homogenate was chemically trapped by o-phenylenediamine and analyzed as the stable phenazine derivative. Tryptamine-4,5-dione localization as adducted proteins in the colorectal tissue was immunohistochemically confirmed, and as demonstrated by both methods, this resu...
Manuka honey is known as one of the premium honeys because of its unique property: a potent antib... more Manuka honey is known as one of the premium honeys because of its unique property: a potent antibacterial activity. Leptosperin, methyl syringate 4-O-β-d-gentiobioside, has been specifically identified in manuka honey. Because leptosperin is relatively stable under warmer conditions, measuring leptosperin levels may be applied to authenticate manuka honey. In this study, an immunochromatographic separation and quantification of leptosperin techniques have been developed. The concentration of leptosperin measured by immunochromatography was significantly correlated with the concentration measured by high-performance liquid chromatography (HPLC) or enzyme-linked immunosorbent assay (ELISA). Because the immunochromatographic method is rapid and reliable, it could be applied to on-site quality control or inspection of honey samples by a beekeeper, a manufacturer, an inspector, a retailer, or a consumer.
Journal of Clinical Biochemistry and Nutrition, 2021
Increased 5-hydroxytryptamine may be associated with the development and progression of inflammat... more Increased 5-hydroxytryptamine may be associated with the development and progression of inflammatory bowel disease. In this study, we examined the suppressive effect of flavonoids on the increased intra- and extracellular 5-hydroxytryptamine levels in rat mast RBL-2H3 cells, known to produce 5-hydroxytryptamine by the phorbol 12-myristate 13-acetate stimulation. Among the flavonoids examined, luteolin and quercetin significantly reduced the cellular 5-hydroxytryptamine concentration. Gene and protein expression analyses revealed that luteolin significantly suppressed cellular tryptophan hydroxylase 1 expression induced by phorbol 12-myristate 13-acetate stimulation. Mitogen-activated protein kinase/extracellular signal-regulated kinase signaling was also suppressed by luteolin, suggesting that this pathway is one of targets of 5-hydroxytryptamine modulation by luteolin. An in vivo experimental colitis model was prepared by administering 2.5% dextran sodium sulfate in drinking water ...
Quercetin is a major flavonoid, present as its glycosidic forms in plant foods. In this study, qu... more Quercetin is a major flavonoid, present as its glycosidic forms in plant foods. In this study, quercetin-3-glucoside (Q3G) was administered intraduodenally to thoracic lymph-cannulated rats, and its lymphatic transport was investigated. The resulting lymphatic and plasma metabolites were identified with LC-MS/MS and compared with those after administration of quercetin aglycone. The total concentration of quercetin metabolites in the lymph was about four times lower than that in the plasma, and quercetin and its methylated form isorhamnetin were detected as their glucuronides, sulfates and diglucuronides both in the lymph and the plasma after Q3G and quercetin administrations. The lymph levels of the glucuronides after Q3G administration were lower than those after quercetin administration, whereas those in the plasma showed the opposite pattern. Both the lymph and plasma levels of the sulfates after Q3G administration were lower than those after quercetin administration. Some of th...
Manuka honey, which shows strong nonperoxide-dependent antibacterial activity, contains unique co... more Manuka honey, which shows strong nonperoxide-dependent antibacterial activity, contains unique components, such as methyl syringate 4-O-β-D-gentiobioside (leptosperin) and its aglycone, methyl syringate (MSYR). To determine the potential for biological activity evoked by the ingestion of leptosperin and MSYR, we investigated the absorption and metabolism of these components in manuka honey. The incubation of MSYR with liver microsomes or S9 fractions in vitro resulted in the formation of MSYR-glucuronide (MSYR-GA), MSYR-sulfate (MSYR-S), and syringic acid as metabolites. Then, manuka honey (15 g) was fed to healthy human volunteers. MSYR-GA, MSYR-S, and MSYR were detected in both plasma and urine. Within plasma, their levels were highest within 0.5 h to 1 h post-ingestion, and most metabolites disappeared within 3 h. In conjunction with the disappearances, a significant amount of metabolites along with trace leptosperin was excreted in urine within 4 h. To elucidate the detailed metabolisms of leptosperin and MSYR, each compound was separately administered to mice. In each case, MSYR-GA, MSYR-S, and MSYR were detected in both plasma and urine. This study shows the major molecular pathway for leptosperin and MSYR metabolism and could facilitate an understanding of biological functions of manuka honey post ingestion.
Serotonin (5-hydroxytryptamine) is a putative substrate for myeloperoxidase, which may convert it... more Serotonin (5-hydroxytryptamine) is a putative substrate for myeloperoxidase, which may convert it into the reactive quinone tryptamine-4,5-dione (TD). In this study, we found that the viability of human SH-SY5Y neuroblastoma cells treated with 25 μM TD was increased to approximately 117%. On the other hand, the cell viability was significantly decreased by exposure to TD (150-200 μM), with an increase in intracellular reactive oxygen species (ROS). Interestingly, pre-treatment of SH-SY5Y cells with 100 μM TD prevented cell death and suppressed intracellular ROS generation evoked by the addition of hydrogen peroxide (H2O2). Expression of the phase-II antioxidant enzyme NAD(P)H: quinone oxidoreductase 1 and haem oxygenase 1 were upregulated by TD at a concentration of 50-100 μM. Nuclear factor erythroid 2-related factor 2 (Nrf2), the regulator of these enzyme, was translocated from the cytosol to the nucleus by 100 μM TD. In summary, moderate concentrations of TD may increase the self...
Journal of clinical biochemistry and nutrition, 2016
Myeloperoxidase is an inflammatory enzyme that generates reactive hypochlorous acid in the presen... more Myeloperoxidase is an inflammatory enzyme that generates reactive hypochlorous acid in the presence of hydrogen peroxide and chloride ion. However, this enzyme also uses bromide ion or thiocyanate as a substrate to form hypobromous or hypothiocyanous acid, respectively. These species play important roles in host defense against the invasion of microorganisms. In contrast, these enzyme products modify biomolecules in hosts during excess inflammation, indicating that the action of myeloperoxidase is both beneficial and harmful. Myeloperoxidase uses other endogenous compounds, such as serotonin, urate, and l-tyrosine, as substrates. This broad-range specificity may have some biological implications. Target molecules of this enzyme and its products vary, including low-molecular weight thiols, proteins, nucleic acids, and lipids. The modified products represent biomarkers of myeloperoxidase action. Moderate inhibition of this enzyme might be critical for the prevention/modulation of exce...
Bioscience, biotechnology, and biochemistry, Jan 7, 2016
Lipid peroxidation products react with cellular molecules, such as DNA bases, to form covalent ad... more Lipid peroxidation products react with cellular molecules, such as DNA bases, to form covalent adducts, which are associated with aging and disease processes. Since lipid peroxidation is a complex process and occurs in multiple stages, there might be yet unknown reaction pathways. Here, we analyzed comprehensively 2'-deoxyguanosine (dG) adducts with oxidized arachidonic acid using liquid chromatography-tandem mass spectrometry and found the formation of 7-(2-oxo-hexyl)-etheno-dG as one of the major unidentified adducts. The formation of this adduct was reproduced in the reaction of dG with 2-octenal and predominantly with 4-oxo-2-octenal (OOE). We also found that other 2-alkenals (with five or more carbons) generate corresponding 4-oxo-2-alkenal-type adducts. Importantly, it was found that transition metals enhanced the oxidation of C4-position of 2-octenal, leading to the formation of OOE-dG adduct. These findings demonstrated a new pathway for the formation of 4-oxo-2-alkenals...
Elimination of celiac-toxic prolamin peptides and proteins is essential for Triticeae products to... more Elimination of celiac-toxic prolamin peptides and proteins is essential for Triticeae products to be gluten-free. Instead of enzymatic hydrolysis, in this study we investigated metal-catalyzed oxidation of two model peptides, QQPFP, and PQPQLPY, together with a hordein isolate from barley (Hordeum vulgare L.). We established a multiple reaction monitoring (MRM) LC-MS method to detect and quantify proline oxidation fragments. In addition to fragmentation, aggregation and side chain modifications were identified, including free thiol loss, carbonyl formation, and dityrosine formation. The immunoreactivity of the oxidized hordein isolate was considerably decreased in all metal-catalyzed oxidation systems. Cleavage of peptides or protein fragments at the numerous proline residues partially accounts for the decrease. Metal-catalyzed oxidation can thus be used in the modification and elimination of celiac-toxic peptides and proteins.
Archives of biochemistry and biophysics, Jan 9, 2015
At the sites of inflammation, hypohalous acids, such as hypochlorous acid and hypobromous acid (H... more At the sites of inflammation, hypohalous acids, such as hypochlorous acid and hypobromous acid (HOBr), are produced by myeloperoxidase. These hypohalous acids rapidly react with the primary amino groups to produce haloamines, which are relatively stable and can diffuse long distances and cross the plasma membrane. In this study, we examined the effects of taurine, the most abundant free amino acid in the leukocyte cytosol, on the hypohalous acid-dependent formation of 8-chloro-2'-deoxyguanosine (8-CldG) and 8-bromo-2'-deoxyguanosine (8-BrdG). The reaction of taurine with HOBr yielded taurine bromamine, which is the most stable among other bromamines of amino acids. Taurine also enhanced the bromination of only dG among the four 2'-deoxynucleosides, whereas it inhibited the 8-CldG formation. The specificity of taurine for the enhanced formation of halogenated dG is completely different from that of nicotine, an enhancer of chlorination. The amount of dibrominated taurine ...
Bioscience, biotechnology, and biochemistry, Jan 4, 2015
Myeloperoxidase (MPO)-generated halogenating molecules, such as hypochlorous acid and hypobromous... more Myeloperoxidase (MPO)-generated halogenating molecules, such as hypochlorous acid and hypobromous acid (HOBr), in inflammatory regions are postulated to contribute to disease progression. In this study, we showed that ergothioneine (EGT), derived from an edible mushroom, inhibited MPO activity as well as the formation of 8-bromo-2'-deoxyguanosine in vitro. The HOBr scavenging effect of EGT is higher than those of ascorbic acid and glutathione. We initially observed that the administration of Coprinus comatus, an edible mushroom containing a high amount of EGT, inhibited the UV-B-induced inflammatory responses and DNA halogenation, suggesting that EGT is a promising anti-inflammatory agent from mushrooms.
Inflammatory bowel diseases, including Crohn’s disease and ulcerative colitis, are chronic inflam... more Inflammatory bowel diseases, including Crohn’s disease and ulcerative colitis, are chronic inflammatory disorders associated with oxidative stress. The intestines produce 5-hydroxytryptamine that may negatively affect disease state under inflammatory conditions when overproduced. 5-Hydroxytryptamine is a substrate for myeloperoxidase and is converted into reactive tryptamine-4,5-dione. Here, an experimental colitis model was established through oral administration of 5% dextran sulfate sodium to ICR mice for 7 days. Furthermore, the formation of tryptamine-4,5-dione in the colorectal mucosa/submucosa and colorectal tissue was analyzed by chemical and immunochemical methodologies. First, free tryptamine-4,5-dione in the homogenate was chemically trapped by o-phenylenediamine and analyzed as the stable phenazine derivative. Tryptamine-4,5-dione localization as adducted proteins in the colorectal tissue was immunohistochemically confirmed, and as demonstrated by both methods, this resu...
Manuka honey is known as one of the premium honeys because of its unique property: a potent antib... more Manuka honey is known as one of the premium honeys because of its unique property: a potent antibacterial activity. Leptosperin, methyl syringate 4-O-β-d-gentiobioside, has been specifically identified in manuka honey. Because leptosperin is relatively stable under warmer conditions, measuring leptosperin levels may be applied to authenticate manuka honey. In this study, an immunochromatographic separation and quantification of leptosperin techniques have been developed. The concentration of leptosperin measured by immunochromatography was significantly correlated with the concentration measured by high-performance liquid chromatography (HPLC) or enzyme-linked immunosorbent assay (ELISA). Because the immunochromatographic method is rapid and reliable, it could be applied to on-site quality control or inspection of honey samples by a beekeeper, a manufacturer, an inspector, a retailer, or a consumer.
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