Michitaka Naito

Flavonoids as Substrates and Inhibitors of Myeloperoxidase: Molecular Actions of Aglycone and Metabolites

Myeloperoxidase (MPO), secreted by activated neutrophils and macrophages at the site of inflammation, may be implicated in the oxidation of protein/lipoprotein during the development of cardiovascular diseases. Flavonoids have been suggested to act as antioxidative and anti-inflammatory agents in vivo; however, their molecular actions have not yet been fully understood. In this study, we examined the molecular basis of the inhibitory effects of dietary flavonoids, such as quercetin, and their metabolites on the catalytic reaction of MPO using a combination of biological assays and theoretical calculation studies. Immunohistochemical staining showed that a quercetin metabolite was colocalized with macrophages, MPO, and dityrosine, an MPO-derived oxidation product of tyrosine, in human atherosclerotic aorta. Quercetin and the plasma metabolites inhibited the formation of dityrosine catalyzed by the MPO enzyme and HL-60 cells in a dose-dependent manner. Spectrometric analysis indicated that quercetin might act as a cosubstrate of MPO resulting in the formation of the oxidized quercetin. Quantitative structure-activity relationship studies showed that the inhibitory actions of flavonoids strongly depended not only on radical scavenging activity but also on hydrophobicity (log P). The requirement of a set of hydroxyl groups at the 3, 5, and 4-positions and C2-C3 double bond was suggested for the inhibitory effect. The binding of quercetin and the metabolites to a hydrophobic region at the entrance to the distal heme pocket of MPO was also proposed by a computer docking simulation. The current study provides the structure-activity relationships for flavonoids as the anti-inflammatory dietary constituents targeting the MPO-derived oxidative reactions in vivo.

(−)-Epicatechin gallate accumulates in foamy macrophages in human atherosclerotic aorta: Implication in the anti-atherosclerotic actions of tea catechins

The localization and target sites of tea catechins underlying their biological activity including anti-atherosclerotic activity have not yet been fully understood. To identify the target sites of catechins in vivo, we have developed a novel monoclonal antibody (mAb5A3) specific for (-)-epicatechin-3-gallate (ECg), one of the major tea catechins. The immunoreactive materials with mAb5A3 were detected in the human atherosclerotic lesions but not in the normal aorta, and were specifically localized in the macrophage-derived foam cells. In vitro experiments using macrophage-like cell lines also showed the significant accumulation of ECg in the cells. We also demonstrated that ECg could suppress the gene expression of a scavenger receptor CD36, a key molecule for foam cell formation, in macrophage cells. These results, for the first time, showed the target site of a tea component ECg in the aorta and might provide a mechanism for the anti-atherosclerotic actions of the catechins.

Lemon Polyphenols Suppress Diet-induced Obesity by Up-Regulation of mRNA Levels of the Enzymes Involved in β-Oxidation in Mouse White Adipose Tissue

The aim of this study was to investigate the effect of dietary lemon polyphenols on high-fat diet-induced obesity in mice, and on the regulation of the expression of the genes involved in lipid metabolism to elucidate the mechanisms. Mice were divided into three groups and fed either a low fat diet (LF) or a high fat diet (HF) or a high fat diet supplemented with 0.5% w/w lemon polyphenols (LP) extracted from lemon peel for 12 weeks. Body weight gain, fat pad accumulation, the development of hyperlipidemia, hyperglycemia, and insulin resistance were significantly suppressed by lemon polyphenols. Supplementation with lemon polyphenols also significantly up-regulated the mRNA level of the peroxisome proliferator activated receptor-α (PPARα) compared to the LF and HF groups in the liver. Furthermore, the mRNA level of acyl-CoA oxidase (ACO) was up-regulated in the LP group compared to the LF group, but not HF group in the liver, and was also significantly increased in the epididymal white adipose tissue. Thus, feeding with lemon polyphenols suppressed body weight gain and body fat accumulation by increasing peroxisomal β-oxidation through up-regulation of the mRNA level of ACO in the liver and white adipose tissue, which was likely mediated via up-regulation of the mRNA levels of PPARα.

Quantification of Modified Tyrosines in Healthy and Diabetic Human Urine using Liquid Chromatography/Tandem Mass Spectrometry.

The quantification of urinary oxidized tyrosines, dityrosine (DiY), nitrotyrosine (NY), bromotyrosine (BrY), and dibromotyrosine (DiBrY), was accomplished by quadruple liquid chromatography-tandem mass spectrometry (LC/MS/MS). The sample was partially purified by solid phase extraction, and was then applied to the LC/MS/MS using multiple-reaction monitoring (MRM) methods. The analysis for the DiY quantification was done first. The residual samples were further butylated with n-butanol/HCl, and the other modified tyrosines were then quantified with isotopic dilution methods. MRM peaks of the modified tyrosines (DiY, NY, BrY, and DiBrY) from human urine were measured and the elution times coincided with the authentic and isotopic standards. The amounts of modified tyrosines in healthy human urine (n = 23) were 8.8 ± 0.6 (DiY), 1.4 ± 0.4 (NY), 3.8 ± 0.3 (BrY), and 0.7 ± 0.1 (DiBrY) µmol/mol of creatinine, respectively. A comparison of the modified tyrosines with urinary 8-oxo-deoxyguanosine, pentosidine, and Nε-(hexanoyl)lysine was also performed. Almost all products, except for NY, showed good correlations with each other. The amounts of the modified tyrosines (NY, BrY, and DiBrY) in the diabetic urine were higher than those in the urine from healthy people.

Delayed postprandial metabolism of triglyceride-rich lipoproteins in obese young men compared to lean young men.

BACKGROUNDnObesity, especially visceral obesity, has been known to affect lipoprotein metabolism, but it is not clear whether obesity in young, apparently healthy men is associated with postprandial triglyceride-rich lipoprotein (TRL) metabolism.nnnMETHODSnTen young normolipidemic, normoglycemic obese men (20.6 ± 0.5 y, BMI 27.5 ± 1.0 kg/m(2)) and 11 lean healthy men (22.1 ± 0.4 y, 21.2 ± 0.4 kg/m(2)) ingested OFTT cream (1g/kg body weight). Fasting and postprandial blood samples were obtained for up to 6h, and serum lipids and lipoproteins were analyzed.nnnRESULTSnThe obese men with a fasting triglyceride (TG) in the normal range and not different from the fasting value of lean controls had a prolonged postprandial response, indicated by a significantly greater incremental areas under the curve in serum TG, TRL-TG, and remnant-like particle-cholesterol (RLP-C) compared with controls. Plasma glucose levels did not change during the test. Differences in serum insulin levels and homeostasis model assessment-insulin resistance (HOMA-IR) were not statistically significant between the two groups; however, trends toward higher levels were shown in obese young men.nnnCONCLUSIONSnThe obese young men showed significantly delayed TRL metabolism compared to the lean young men after fat loading, even though the obese men were normolipidemic. These results suggest the possibility that early insulin resistance in the obese young men may have caused the decrease of lipoprotein lipase activity and induced delayed TRL metabolism. A fat loading test without carbohydrate may provide a useful tool for the detection of delayed postprandial TRL metabolism and early insulin resistance.

Chemical and immunochemical identification of propanoyllysine derived from oxidized n-3 polyunsaturated fatty acid.

It is known that n-3 polyunsaturated fatty acids (PUFAs), such as docosahexaenoic acid and eicosapentaenoic acid, are rapidly oxidized in vitro. Nvarepsilon-(propanoyl)lysine (propionyllysine, or PRL) is formed from the reaction of the oxidized products of n-3 PUFAs and lysine. To evaluate the oxidized n-3 PUFA-derived protein modifications in vivo, we have developed detection methods using a novel monoclonal antibody against PRL as well as liquid chromatography-mass spectrometry (LC/MS/MS). The antibody obtained specifically recognized PRL. A strong positive staining in atherosclerotic lesions of hypercholesterolemic rabbits was observed. We have also simultaneously identified and quantified both urinary PRL and urinary Nvarepsilon-(hexanoyl)lysine, using LC/MS/MS using isotope dilution methods. The level of urinary PRL (21.6+/-10.6 micromol/mol of creatinine) significantly correlated with the other oxidative stress markers, 8-oxo-deoxyguanosine, dityrosine, and isoprostanes. The increase in the excretion of amide adducts into the urine of diabetic patients was also confirmed compared to healthy subjects. These results suggest that PRL may be good marker for n-3 PUFA-derived oxidative stress in vivo.

Plasma fibrinogen and its association with cardiovascular risk factors in apparently healthy Japanese subjects

Recent evidence has shown the association of increased plasma fibrinogen levels with subsequent coronary heart disease or stroke. Fibrinogen is an acute-phase inflammatory reactant as well as a clotting factor. The authors investigated an association between fibrinogen levels and cardiovascular risk factors in apparently healthy Japanese subjects, while considering C-reactive protein (CRP) levels, a marker of the inflammatory status. Plasma fibrinogen and serum CRP from 2u2009706 participants in an annual mass screening examination, held in Matsukawa, Nagano, Japan were measured. A total of 2u2009355 subjects (816 men and 1u2009539 women) were analyzed after excluding individuals with a history of diabetes mellitus, heart disease, or stroke. Plasma fibrinogen was strongly correlated with CRP levels. After adjusting the CRP levels, fibrinogen was positively associated with age, smoking status, total cholesterol, and hemoglobin A1c (HbA1c) in men, and with age, total cholesterol, and HbA1c in women. On the other hand, high-density lipoprotein (HDL) cholesterol was a strong negative correlate of fibrinogen in both genders. Fibrinogen levels also tended to be associated positively with body mass index in both genders and negatively with exercise habits in men. The present multiple regression analysis has shown that plasma fibrinogen levels are correlated with conventional cardiovascular risk factors even after adjusting for the CRP levels. Persons with cardiovascular risk factors tended to have higher fibrinogen levels, suggesting that all elevated plasma fibrinogen concentration in those with risk factors may further increase the risk of the development of atherothrombosis and subsequent cardiovascular disease through the blood coagulation system.

The ingestion of a fructose-containing beverage combined with fat cream exacerbates postprandial lipidemia in young healthy women.

AIMnTo investigate the acute effects of the ingestion of a fructose-containing beverage combined with fat on postprandial lipoprotein metabolism.nnnMETHODSnTwelve young healthy Japanese women with apolipoprotein E phenotype 3/3 were enrolled in this study. At each of four sessions, the subjects ingested one of four sugar beverages containing fructose and/or glucose (total: 0.5 g/kg body weight) combined with OFTT cream (1 g/kg, 0.35 g/kg as fat) in a randomized crossover design. The four sugar beverages were as follows: 100% (w/w) fructose (F100), 90% fructose + 10% glucose (F90G10), 55% fructose + 45% glucose (F55G45) and 100% glucose (G100). Venous blood samples were obtained at baseline and 0.5, one, two, four and six hours after ingestion.nnnRESULTSnThe serum concentrations of TG in the F100, F90G10 and F55G45 trials were significantly higher than each fasting value at two and four hours, and returned to baseline at six hours, except in the F100 trial. The concentrations at four hours and the incremental areas under the curve for the hepatic triglyceride-rich lipoprotein-triglyceride (VLDL-TG(TM)) levels in the F100 and F90G10 trials were significantly higher and larger, respectively, than those observed in the G100 trial. Meanwhile, the concentrations of RLP-TG and apolipoprotein B-48 peaked at two hours in the G100 trial, versus four hours in the other trials, and did not return to baseline at six hours, except in the G100 trial. At four hours, the ⊿apoB48 tended to be higher in the F100 trial than in the G100 trial.nnnCONCLUSIONSnThe ingestion of a high-fructose-containing beverage with fat cream delays the clearance of chylomicron and its remnant derived from the intestine and enhances the secretion of triglyceride-rich lipoprotein particles from the liver, thereby inducing postprandial lipidemia, even in young healthy women.

Immunochemical detection of flavonoid glycosides: Development, specificity, and application of novel monoclonal antibodies

Flavonoid-rich diets are expected to decrease the risk of cardiovascular diseases. The localization and target sites of flavonoids underlying the protective mechanism in vivo have not been fully investigated because the methods for detection of flavonoids have been limited to chemical analysis such as high-performance liquid chromatography. To further understand the actions of flavonoids in vivo, we developed a novel methodology that immunochemically evaluates flavonoids using specific antibodies. Quercetin-3-glucuronide (Q3GA), a major metabolite in human plasma, was coupled with keyhole limpet hemocyanin. Alternatively, the sugar moiety of quercetin-3-glucoside (Q3G) was succinylated and then coupled with a carrier protein. Using these two immunogens, we finally obtained two monoclonal antibodies, mAb14A2 and mAb11G6, from the immunogen using Q3GA and Q3G, respectively. Competitive enzyme-linked immunosorbent assay showed the unique difference in the specificity between the two similar antibodies: mAb14A2 recognized several quercetin-3-glycosides including Q3G and rutin but mAb11G6 was highly specific to the Q3G structure. The macrophage-derived foam cells in human atherosclerotic lesions were significantly stained with mAb14A2 but scarcely with mAb11G6. These results showed that the anti-flavonoid glycoside antibodies are useful tools for evaluating their localization in tissues and that the specificities strongly depend on the immunogen design for synthesizing the hapten-protein conjugates.

A novel quinone derived from 5-hydroxyindoleacetic acid reacts with protein: Possible participation of oxidation of serotonin and its metabolite in the development of atherosclerosis

The modification of 5-hydroxyindoleacetic acid (5HIAA) by myeloperoxidase with a xanthine oxidase system was investigated by chromatographic analyses. Two major products were identified as a dimer and quinone (indoleacetate dione) of 5HIAA. The formation of a quinone moiety was also confirmed by chemical trapping with o-phenylenediamine. In the presence of N-acetyl-cysteine (NAC), a quinone-NAC adduct was formed. When glyceraldehyde 3-phosphate dehydrogenase was exposed to the myeloperoxidase system with 5HIAA, quinone adducts were formed on the protein molecule. A monoclonal antibody was prepared using a quinone-modified protein as an immunogen to immunochemically detect the quinone on a protein. The established antibody recognized the quinone-NAC adduct, quinone-modified poly-L-lysine, and quinone-modified low-density lipoprotein. Quinone-modified proteins in human atherosclerotic lesions were immunohistochemically observed using the established antibody to the quinone and also a monoclonal antibody to tryptamine dione-modified protein, suggesting an occurrence of in vivo oxidation of serotonin and 5HIAA, accompanied by covalent adduction to biomolecules.