Although uric acid is known to react with many reactive oxygen species, its specific oxidation products have not been fully characterized. We now report that 5-N-carboxyimino-6-N-chloroaminopyrimidine-2,4(3H)-dione (CCPD) is a hypochlorite (ClO⁻)-specific oxidation product of uric acid. The yield of CCPD was 40–70% regardless of the rate of mixing of ClO⁻ with uric acid. A previously reported product, allantoin (AL), was a minor product. Its yield (0–20%) decreased with decreasing rate of mixing of ClO⁻ with uric acid, indicating that allantoin is less important in vivo. Kinetic studies revealed that the formation of CCPD required two molecules of ClO⁻ per uric acid reacted. The identity of CCPD was determined from its molecular formula (C₅H₃ClN₄O₄) measured by LC/time-of-flight mass spectrometry and a plausible reaction mechanism. This assumption was verified by the fact that all mass fragments (m/z −173, −138, −113, and −110) fit with the chemical structure of CCPD and its tautomers. Isolated CCPD was stable at pH 6.0–8.0 at 37°C for at least 6 h. The above results and the fact that uric acid is widely distributed in the human body at relatively high concentrations indicate that CCPD is a good marker of ClO⁻ generation in vivo.

This study examines oxidizability in individual aqueous solutions of oleic acid, linoleic acid, α-linolenic acid, γ-linolenic acid and arachidonic acid, and in their mixtures. We used electron spin resonance (ESR), high performance liquid chromatography-electron spin resonance (HPLC-ESR) and high performance liquid chromatography-electron spin resonance-mass spectrometries (HPLC-ESR-MS). We detected 4-carboxybutyl radical derived from γ-linolenic acid, ethyl and 7-carboxyheptyl radicals derived from α-linolenic acid, and pentyl and 7-carboxyheptyl radicals derived from linoleic acid. HPLC-ESR analyses for the individual aqueous solutions of linoleic acid, α-linolenic acid, γ-linolenic acid and arachidonic acid showed less radical form for polyunsaturated fatty acids with more double bonds. On the other hand, HPLC-ESR peak height of 4-carboxybutyl radical, which form through hydrogen atom abstraction at the carbon close to the carboxy end, increased for linoleic acid/γ-linolenic acid, α-linolenic acid/γ-linolenic acid, and γ-linolenic acid/oleic acid mixtures compared to before mixing. Conversely, HPLC-ESR peak heights of ethyl, 7-carboxyheptyl and pentyl radicals, which form through hydrogen atom abstraction at the carbons close to the methyl end, decreased for linoleic acid/α-linolenic acid, linoleic acid/γ-linolenic acid, linoleic acid/oleic acid, linoleic acid/arachidonic acid, α-linolenic acid/γ-linolenic acid, and α-linolenic acid/oleic acid mixtures compared to before mixing.

The generation of localized hydroxyl radical (^•OH) in aqueous samples by low linear energy transfer irradiation was investigated. Several concentrations of 5,5-dimethyl-1-pyrroline-N-oxid solution (from 0.5 to 1,680 mmol/L) were prepared and irradiated with an identical dose of X-ray or γ-ray. The density of ^•OH generation in aqueous solution was evaluated by the electron paramagnetic resonance spin-trapping technique using 5,5-dimethyl-1-pyrroline-N-oxid as an electron paramagnetic resonance spin-trapping agent. The relationship between the molecular density of 5,5-dimethyl-1-pyrroline-N-oxid in the samples and the concentration of 5,5-dimethyl-1-pyrroline-N-oxid-OH generated in the irradiated samples was analyzed. Two different characteristic linear trends were observed in the 5,5-dimethyl-1-pyrroline-N-oxid-OH/5,5-dimethyl-1-pyrroline-N-oxid plots, which suggested ^•OH generation in two fashions, i.e., mmol/L- and mol/L-level local concentrations. The dose, dose rate, and/or the energy of photon irradiation did not affect the shapes of the 5,5-dimethyl-1-pyrroline-N-oxid-OH/5,5-dimethyl-1-pyrroline-N-oxid plots. Moreover, the addition of 5 mmol/L caffeine could cancel the contribution of mmol/L-level ^•OH generation, leaving a trace of mol/L-level ^•OH generation. Thus, the localized mmol/L- and mol/L-level generations of ^•OH, which were independent of experimental parameters such as dose, dose rate, and/or the energy of photon of low linear energy transfer radiation, were established.

Plant sterols are used as food additives to reduce intestinal cholesterol absorption. They also increase fecal neutral sterol (FNS) excretion irrespective of the absorption inhibition. Intestine-mediated reverse cholesterol transport, or trans-intestinal cholesterol efflux (TICE), provides the major part of the increase of FNS excretion. However, it is unknown whether plant sterols stimulate TICE or not. We have shown previously that TICE can be evaluated by brush border membrane (BBM)-to-lumen cholesterol efflux. Thus, we examined whether luminal plant sterols stimulate BBM-to-lumen cholesterol efflux in the intestinal tract or not in mice. Cannulated upper jejunum that had been pre-labeled with orally given ³H-cholesterol, was flushed and perfused to collect ³H-cholesterol effluxed back into the lumen from the BBM to estimate the efflux efficiency. Adding 0.5 mg/ml of plant sterols, but not cholesterol, in the perfusion solution doubled the efflux. Plant sterols enter the BBM and are effluxed back to the lumen rapidly, in which process cholesterol transporters in the BBM are involved. We thus speculate that phytosterols alter cholesterol flux in the BBM; thereby, increases BBM-to-lumen cholesterol efflux, resulting in the increased TICE.

Dipeptidyl peptidase-4 (DPP-4) is a critical molecule for the metabolism of incretins. In addition, DPP-4 is known as CD26, the receptor of T cells, and plays important role in activation of T cells. Recently, DPP-4 inhibitors (DPP4i) are reported to have several immunologic effects beyond glycemic control. DPP4i seem to have anti-inflammatory effects in patients with type 2 diabetes. This might be direct effects on T cells. However, the close mechanism is not clear. To evaluate the possibility, we performed ex vivo assays by using primarily human CD4⁺ T cells (CD4) and CD8⁺ T cells (CD8). We purified primary naïve CD4 and CD8 from human peripheral blood. Then, we evaluated the effect of DPP4i on the proliferation of naïve T cells and the cytokine production in ex vivo experiments. The proliferation of CD4 and CD8 were suppressed by adding DPP4i in a dose dependent manner. However, DPP4i did not inhibit cytokine production from CD4. It was revealed by phospho-flow that the T cell receptor (TCR) signaling was attenuated in the presence of DPP4i. Taken together, DPP4i modulated TCR signaling, which contributed to attenuate the proliferation of CD4 and CD8. DPP4i have adverse effects for the proliferation of human T cells.

Dietary intervention for preventing postprandial increases in glucose level by replacing high-glycemic index (GI) carbohydrates with lower-GI carbohydrate has been proposed as a strategy for treating insulin-resistant metabolic disorders such as type II diabetes. In this study, we examined the effect of short-term replacement of starch with a low-GI disaccharide, isomaltulose, on insulin action in skeletal muscle. Male Wistar rats were fed isomaltulose for 12 h during their dark cycle. In isolated epitrochlearis muscle, insulin-induced glucose uptake was greater in tissue from rats treated with isomaltulose than from those treated with starch. This insulin-sensitizing effect occurred independently of changes visceral fat mass. To determine whether this sensitization was specific to insulin stimulation, we also measured glucose uptake in response to exercise. In isolated epitrochlearis muscles from rats that performed swimming exercise, exercise-induced glucose uptake was higher in isomaltulose-treated than starch-treated animals. This amplification was associated with increased phosphorylation of exercise-induced AMP-activated protein kinase. In conclusion, our results demonstrate that short-term replacement of starch with isomaltulose enhances both insulin-dependent and -independent glucose uptake in isolated skeletal muscle. This transient replacement of carbohydrate with isomaltulose, together with exercise, represents a potentially effective approach for the management of insulin resistance.

The exact mechanisms of hepatocellular carcinoma development in non-alcoholic steatohepatitis remain unclear. In this study, we used a new class of high-fat diet, which could induce hepatocellular carcinoma development without the use of general chemical carcinogens or knockout mice. We investigated the correlation between hepatocellular carcinoma and oxidative stress/anti-oxidant effects after depletion of the gut microbiota by treatment with antibiotics. Mice fed with the steatohepatitis-inducing high-fat diet (STHD-01) for 41 weeks developed hepatocellular carcinoma. Antibiotic-treatment in mice fed with STHD-01 significantly depleted the gut microbiota and significantly ameliorated liver injury/histology. The tumor numbers of hepatocellular carcinoma were dramatically decreased by the antibiotics-treatment. We analyzed the factors involved in oxidative stress and anti-oxidant effects. Oxidative stress was elevated in mice fed with STHD-01, whereas some anti-oxidant factors were significantly elevated after antibiotics treatment. These results suggest that the gut microbiota is a key factor in improving oxidative stress induced by STHD-01 feeding.

Aging populations are expanding worldwide, and the increasing requirement for nursing care has become a serious problem. Furthermore, successful aging is one of the highest priorities for individuals and societies. Centenarians are an informative cohort to study and inflammation has been found to be a key factor in predicting cognition and physical capabilities. Inflammation scores have been determined based on the levels of cytokines and C-reactive protein, however, serum antioxidants and lipid profiles have not been carefully examined. We found that the redox balance of coenzyme Q10 significantly shifted to the oxidized form and levels of strong antioxidants, such as ascorbic acid and unconjugated bilirubin, decreased significantly compared to 76-year-old controls, indicating an increased oxidative stress in centenarians. Levels of uric acid, an endogenous peroxynitrite scavenger, remained unchanged, suggesting that centenarians were experiencing moderate, chronic inflammatory conditions. Centenarians exhibited a hypocholesterolemic condition, while an increase in the ratio of free cholesterol to cholesterol esters suggests some impairment of liver function. Serum free fatty acids and monoenoic acid composition, markers of tissue oxidative damage, were significantly decreased in centenarians, indicating an impairment in the tissue repair system. Despite an elevation of the coenzyme Q10 binding protein Psap, serum total coenzyme Q10 levels decreased in centenarians. This suggests a serious deficiency of coenzyme Q10 in tissues, since tissue levels of coenzyme Q10 significantly decrease with age. Therefore, coenzyme Q10 supplementation could be beneficial for centenarians.

Sepsis remains one of the leading causes of death in intensive care units. The early phase of sepsis is characterized by a massive formation of reactive oxygen and nitrogen species such as superoxide and nitric oxide. However, few comprehensive studies on plasma antioxidants have been reported. Increased oxidative stress was confirmed in sepsis patients (n = 18) at the time of hospitalization by a significant decrease in plasma ascorbic acid and a significant increase in the percentage of oxidized form of coenzyme Q10 in total coenzyme Q10 compared to age-matched healthy controls (n = 62). Tissue oxidative damage in patients was suggested by a significant decrease in polyunsaturated fatty acid contents and a significant increase in oleic acid contents in total free fatty acids. Thus, it is reasonable that plasma uric acid (end product of purines) would be significantly elevated. However, uric acid levels were continuously decreased during hospitalization for 7 days, indicating a continuous formation of peroxynitrite. A greater decrease in free cholesterol (FC) compared to cholesterol esters (CE) was observed. Thus, the FC/CE ratio significantly increased, suggesting deficiency of lecithin-cholesterol acyltransferase secreted from the liver. Plasma levels of prosaposin, a coenzyme Q10 binding protein, significantly decreased as compared to healthy controls. This may be correlated with renal injury in sepsis patients, since the kidney is thought to be a major secretor of prosaposin.

The aim of our study was to examine gender differences of LDL- and HDL-cholesterol subfractions in patients after the acute ischemic stroke with focus on small LDL and HDL subfractions, and their association with oxidative stress markers. In addition, we have monitored the 7-day effect of cholesterol-lowering drugs administered to patients after the acute ischemic stroke, on these subfractions. Eighty two stroke patients and 81 age matched controls were included in this study. Blood was collected from patients within 24 h after the stroke (group A) and re-examined at the 7-day follow-up (group B). We have found gender differences in LDL- and HDL-subfractions in stroke patients, lipid-lowering drugs administered to acute ischemic stroke patients significantly reduced all measured parameters of lipoprotein profile. In the group A LDL1 subfraction positively correlated with activity of antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase) indicating a protective role of this subfraction. On the contrary, small HDL subfractions positively correlated with lipoperoxide levels and negatively with trolox equivalent antioxidant capacity in plasma suggesting a negative role of these subfractions. In this work we have confirmed the hypothesis of atherogenic properties of small HDL subfractions and anti-atherogenic properties of large LDL1-subfractions.

A large proportion of patients demonstrating obscure gastrointestinal bleeding (OGIB) are antithrombotic users and need to undergo small bowel capsule endoscopy (SBCE). We examined the effect of discontinuation of antithrombotics on the diagnostic yield of SBCE. Additionally, we assessed predictive factors associated with positive SBCE findings. Our study included 130 patients using antithrombotics who underwent SBCE for overt OGIB. The primary endpoint was the difference in the rate of positive SBCE findings between patients who continued and those who discontinued antithrombotics. Secondary endpoints were to investigate the effect of discontinuation of antithrombotics using a propensity score analysis, and to assess predictive factors associated with a positive SBCE. Among the 73 patients who continued use of antithrombotics, 36 (49.3%) patients demonstrated positive findings, while among the 57 patients who discontinued antithrombotics, 35 (61.4%) patients showed positive findings. Rates of positive SBCE findings didn’t differ between the two groups. After we performed propensity score matching, discontinuation didn’t affect the rate of positive SBCE findings. The lowest hemoglobin level was the only independent predictive factor associated with positive SBCE findings. In conclusion, discontinuation of antithrombotic therapy didn’t affect the diagnostic yield of SBCE in patients presenting with overt OGIB.

To investigate sex differences in the associations among metabolic syndrome, obesity, adipose tissue-related biomarkers, and colorectal adenomatous polyps, a cross-sectional, multicenter study was conducted on 489 consecutive individuals who underwent their first colonoscopy at 3 hospitals. Plasma concentrations of adiponectin and leptin, as well as homeostatic model assessment of insulin resistance were also evaluated. The presence and number of adenomatous polyps, including advanced adenoma, were higher in men than in women. Metabolic syndrome was a risk factor for adenomatous polyps in both sexes. Large waist circumference was an independent risk factor for adenomatous polyps in men, and high BMI and large waist circumference were risk factors for adenomatous polyps in women. Interestingly, low BMI was associated with large adenomatous polyps (≥10 mm) and advanced adenoma, and waist-hip ratio was involved in proximal adenomatous polyp development only in women. In contrast, the highest quartile of leptin concentration had a 3.67-fold increased adenomatous polyp risk compared with the lowest quartile only in men. These results indicate that regarding colorectal pathogenesis, sex differences were identified in obesity but not in metabolic syndrome. Visceral obesity and a high serum leptin level may be risk factors for colorectal adenomatous polyp development in Japanese men.

The aim of this study was to assess the perioperative invasiveness of endoscopic submucosal dissection for colorectal cancer quantitatively by using energy metabolism. In fifty-three patients who underwent endoscopic submucosal dissection for colorectal cancer, resting energy expenditure using an indirect calorimeter, body weight and basal energy expenditure using the Harris–Benedict equation before and after endoscopic submucosal dissection. Resting energy expenditure/body weight and resting energy expenditure/basal energy expenditure were 19.7 ± 2.5 kcal/kg/day and 0.96 ± 0.12 on the day of endoscopic submucosal dissection, whereas one day after the endoscopic submucosal dissection they increased to 21.0 ± 2.9 kcal/kg/day and 1.00 ± 0.13 (p<0.001 and p<0.05, respectively). The stress factor on the postoperative day 1 was computed as 1.06. The increase was lower comparing with that experienced for surgery, suggesting that the perioperative invasiveness of colorectal endoscopic submucosal dissection is lower in comparison to that during surgery. Furthermore, in spite of technical difficulty, stress factor of colorectal endoscopic submucosal dissection was approximately equal to that of gastric endoscopic submucosal dissection. (The study of the resting energy metabolism and stress factor using an indirect calorimeter in the perioperative period of endoscopic operation: UMIN000027135)