Daily hydroxyl radical scavenging capacity of mammals.

Both the formation and reactions of hydroxyl radical (•OH) are quantitative chemical reactions even in mammalians, and so we can reproduce such in vivo reactions in test tubes. Daily urinary excretions of some reaction products have been used to estimate the amount of •OH produced daily. Although urinary 8-hydroxydeoxyguanosine (8-OHdG) is a well-known marker of •OH, we have shown that creatol (CTL: 5-hydroxycreatinine), an •OH adduct of creatinine (Crn), and its metabolite, methylguanidine (MG), are better markers, because the amount of •OH scavenged by deoxyguanosine (dG) in the body is negligible. We measured CTL and MG together with Crn in 24-h urine, and calculated their molar sum, CTL + MG, providing a daily estimate of moles of •OH scavenged with Crn, and, from the molar ratio (CTL + MG)/Crn, we can calculate the percentage of Crn that was used to scavenge •OH. Healthy subjects and normal rats were indicated to use circa (ca.) 0.2 and 0.3% of Crn in order to scavenge •OH, respectively, because the corresponding ratios, scavenged •OH/Crn, were 2.2 and 3.0 mmole/mole (24-h urine) (Crn scavenged ca. 20-25 µmole and ca. 200 pmole of •OH in healthy subjects and normal rats, respectively). Since 8-OHdG/Crn has been reported to be 1.9 µmole/mole (24-h urine), the daily scavenging capacity with Crn is 10(3)-fold more than dG. In patients with chronic renal failure (CRF) or chronic kidney disease (CKD) at stages 3-5: glomerular filtration rate (GFR) < 60 mL/min/1.73 m(2), •OH levels increased in proportion to the severity of CKD: up to ca. 3% of Crn was used daily in order to scavenge •OH. Although the accumulation of MG in organs has not been reported except for the brain and skin tissues in normal animals, •OH increases markedly and MG becomes detectable in all organs such as the kidney, liver, and heart in CRF rats.

A whole-grain-rich diet reduces urinary excretion of markers of protein catabolism and gut microbiota metabolism in healthy men after one week.

Epidemiological studies consistently find that diets rich in whole-grain (WG) cereals lead to decreased risk of disease compared with refined grain (RG)-based diets. Aside from a greater amount of fiber and micronutrients, possible mechanisms for why WGs may be beneficial for health remain speculative. In an exploratory, randomized, researcher-blinded, crossover trial, we measured metabolic profile differences between healthy participants eating a diet based on WGs compared with a diet based on RGs. Seventeen healthy adult participants (11 female, 6 male) consumed a controlled diet based on either WG-rich or RG-rich foods for 2 wk, followed by the other diet after a 5-wk washout period. Both diets were the same except for the use of WG (150 g/d) or RG foods. The metabolic profiles of plasma, urine, and fecal water were measured using (1)H-nuclear magnetic resonance spectroscopy and gas chromatography-mass spectrometry (plasma only). After 1 wk of intervention, the WG diet led to decreases in urinary excretion of metabolites related to protein catabolism (urea, methylguanadine), lipid (carnitine and acylcarnitines) and gut microbial (4-hydroxyphenylacetate, trimethylacetate, dimethylacetate) metabolism in men compared with the same time point during the RG intervention. There were no differences between the interventions after 2 wk. Urinary urea, carnitine, and acylcarnitine were lower at wk 1 of the WG intervention relative to the RG intervention in all participants. Fecal water short-chain fatty acids acetate and butyrate were relatively greater after the WG diet compared to the RG diet. Although based on a small population and for a short time period, these observations suggest that a WG diet may affect protein metabolism.

NMR-based metabolomics study of canine bladder cancer.

Bladder cancer is one of the leading lethal cancers worldwide. With the high risk of recurrence for bladder cancer following the initial diagnoses, lifelong monitoring of patients is necessary. The lack of adequate sensitivity and specificity of current noninvasive monitoring approaches including urine cytology, other urine tests, and imaging, underlines the importance of studies that focus on the detection of more reliable biomarkers for this cancer. The emerging area of metabolomics, which deals with the analysis of a large number of small molecules in a single step, promises immense potential for discovering metabolite markers for screening and monitoring treatment response and recurrence in patients with bladder cancer. Since naturally-occurring canine transitional cell carcinoma of the urinary bladder is very similar to human invasive bladder cancer, spontaneous canine transitional cell carcinoma has been applied as a relevant animal model of human invasive transitional cell carcinoma. In this study, we have focused on profiling the metabolites in urine from dogs with transitional cell carcinoma and healthy control dogs combining nuclear magnetic resonance spectroscopy and statistical analysis methods. (1)H NMR-based metabolite profiling analysis was shown to be an effective approach for differentiating samples from dogs with transitional cell carcinoma and healthy controls based on a partial least square-discriminant analysis of the NMR spectra. In addition, there were significant differences in the levels of six individual metabolites between samples from dogs with transitional cell carcinoma and the control group based on the Student's t-test. These metabolites were selected to build a separate partial least square-discriminant analysis model that was then used to test the classification accuracy. The result showed good classification between transitional cell carcinoma and control groups with the area under the receiver operating characteristic curve of 0.85. The sensitivity and specificity of the model were 86% and 78%, respectively. These results suggest that urine metabolic profiling may have potential for early detection of bladder cancer and of bladder cancer recurrence following treatment, and may enhance our understanding of the mechanisms involved.

First indications demonstrating the preventive effects of NZ-419, a novel intrinsic antioxidant, on the initiation and/or progression of chronic renal failure in rats.

The concentration of NZ-419 (5-hydroxy-1-methylimidazolidine-2,4-dione), an intrinsic antioxidant, has been shown to increase in the sera of animals and patients with chronic renal failure (CRF). This is the first report that orally administered exogenous NZ-419 prevents the initiation and/or progression of CRF in rats using an adenine-loaded model. After 24 d of adenine loading, there was a ca. 90% decrease in creatinine clearance (C(Cr)) in the control rats. Treatment with NZ-419 from the beginning significantly inhibited the decrease in C(Cr) and also the increase in serum creatinine (sCr). Bio-markers for in vivo hydroxyl radicals, the serum methylguanidine (sMG) level, and sMG/sCr molar ratio, not only in serum but also in the urine, kidney, liver, and muscle indicated that NZ-419 inhibited the increase in oxidative stress induced by CRF in rats. An increase of guanidinosuccinic acid, an another bio-marker of oxidative stress, was also inhibited with NZ-419.

Urinary excretion of creatol, an in vivo biomarker of hydroxyl radical, in patients with chronic renal failure.

Creatol (CTL) is a hydroxyl radical adduct of creatinine (Cr). The serum methylguanidine (MG) level and the MG/Cr molar ratio are reported to be biomarkers for oxidative stress. The aim of this study was to examine whether urinary excretion of CTL, another oxidative stress-related marker, is increased in patients with chronic renal failure (CRF). One hundred twenty-four non-dialyzed patients with chronic renal failure (serum Cr level, 1.3-10.0 mg/dL) were recruited from our hospitals. Urine and serum levels of CTL and MG were determined by high-performance liquid chromatography with the use of 9, 10- phenanthrenequinone as a fluorogenic reagent. The CTL/Cr and (CTL+MG)/Cr molar ratios in spot urine samples were also compared with those in 24-h urine samples. The urinary CTL/Cr and (CTL+MG)/Cr molar ratios increased with decreases in Cr clearance in patients with CRF. Correlations between serum and spot urine (CTL+MG)/Cr and between serum and spot urine CTL/Cr were quite similar to those in 24-h urine samples. CTL/Cr and (CTL+MG)/Cr molar ratios in both 24-h urine and spot urine samples appear to be useful indices of the severity of CRF.

Comparison of hydroxyl radical generation in patients undergoing coronary artery bypass grafting with and without cardiopulmonary bypass.

We measured the hydroxyl radical (.OH) generation in fourteen patients undergoing coronary artery bypass grafting (CABG), of whom seven patients underwent on-pump CABG with cardiopulmonary bypass (CPB) and seven patients underwent off-pump CABG without CPB. To detect .OH generation, we measured the urinary excretion of .OH products of creatinine (Cr), creatol (CTL; 5-hydroxycreatinine) and methylguanidine (MG) with HPLC using the one point sampling and collected urine during and after the operation. The urinary CTL value corrected urinary Cr value of on-pump CABG significantly increased about 3-5 times from the beginning of CPB to 4 h after operation compared to the baseline value before CPB in both the collected urine and the one point sampling urine. The urinary MG/Cr value in both groups did not change significantly. Significantly increased .OH generation was found during and soon after on-pump CABG.

Influence of 72% injury in one kidney on several organs involved in guanidino compound metabolism: a time course study.

Arginine (Arg) produced from citrulline originates mostly from kidneys. Arg is involved in guanidino compound biosynthesis, which requires interorgan co-operation. In renal insufficiency, citrulline accumulates in the plasma in proportion to renal damage. Thus, disturbances in Arg and guanidino compound metabolism are expected in several tissues. An original use of the model of nephrectomy based on ligating branches of the renal artery allowed us to investigate Arg and guanidino compound metabolism simultaneously in injured (left) and healthy (right) kidneys. The left kidney of adult rats was subjected to 72% nephrectomy. Non-operated, sham-operated and nephrectomized rats were studied for a period of 21 days. Constant renal growth was observed only in the healthy kidneys. Guanidino compound levels were modified transiently during the first 48 h. The metabolism and/or tissue content of several guanidino compounds were disturbed throughout the experimental period. Arg synthesis was greatly reduced in the injured kidney, while it increased in the healthy kidney. The renal production of guanidinoacetic acid decreased in the injured kidney and its urinary excretion was reduced. The experimentally proven toxins alpha-keto-delta-guanidinovaleric acid and guanidinosuccinic acid (GSA) accumulated only in the injured kidney. The urinary excretion of GSA and methylguanidine increased in nephrectomized rats. When the injured kidney grew again, the level of some guanidino compounds tended to normalize. Nephrectomy affected the guanidino compound levels and metabolism in muscles and liver. In conclusion, the specific accumulation of toxic guanidino compounds in the injured kidney reflects disturbances in renal metabolism and function. The healthy kidney compensates for the injured kidney's loss of metabolic functions (e.g. Arg: production). This model is excellent for investigating renal metabolism when a disease destroys a limited area in one kidney, as is observed in patients.

[Clinical significance of urinary methylguanidine in non-dialyzed patients with chronic renal failure--prediction of progression rate of renal dysfunction].

To clarify a clinical significance of urinary methylguanidine (U-MG, micrograms/dl) in non-dialyzed patients with chronic renal failure, we measured U-MG, urinary creatinine (U-Cr, mg/dl) and serum creatinine (S-Cr, mg/dl), concurrently and continually in 36 out-patients whose S-Cr was over 4.0 mg/dl. Fresh urine sample was obtained and U-MG was measured by an enzymic method. S-Cr concentrations (mean +/- SD) at the initiation of dialysis therapy were comparable in non-diabetics and in diabetics, being 13.4 +/- 4.54mg/dl and 8.23 +/- 1.96mg/dl, respectively, with statistical significance (p < 0.01). U-MG/Cr (micrograms/mg. Cr) values were also different between them, being 9.28 +/- 4.97 in non-diabetics and 5.19 +/- 1.29 in diabetics, but with lesser statistical significance (p < 0.05) than S-Cr. U-MG/Cr correlated well with the terms till the initiation of dialysis therapy (r = 0.7189, p < 0.001), more significantly than S-Cr (r = 0.5506, p < 0.001) in parabolic regression analysis. U-MG/Cr were greatly varied among the 7 patients, although whose S-Cr was nearly the same level (6.0-6.9mg/dl). Most importantly, the tendency was found in these patients that the higher U-MG/Cr, the earlier the renal replacement therapy instituted. These results suggests that U-MG/Cr can be reliable parameter to predict the progression rate of renal failure and to help to know when renal replacement therapy should be instituted.

[Urinary excretion rate of methylguanidine as a new maker of active oxygen in vivo: demonstration in hyperbaric oxygen therapy].

Methylguanidine (MG) which is known as a uremic toxin, is synthesized from creatinine (Cre). We have clarified that active oxygen plays an important role on MG synthesis in vitro and in rat hepatocytes. On the other hand, hyperoxia is very injurious in various tissues, and it has been reported that active oxygen produced in hyperoxia plays an important role on the tissue injury. This study was performed to investigate the effect of hyperoxia on MG synthesis in vivo. The subjects in this study were patients who were treated by hyperbaric oxygen therapy (HBO). Serum Cre, MG, and urinary Cre, MG before and after HBO were measured in these subjects. The subjects were classified into four groups. Group I-III were undergone HBO with condition of 100% O2, 2 atmosphere absolute (ATA), 1 hour, (I: Ccr less than 10 ml/min, II: 10 less than or equal to Ccr less than 50 ml/min, III: Ccr greater than or equal to 50 ml/min) and group IV (Ccr greater than or equal to 50 ml/min) with 100%O2, 3ATA, 1 hour. Urinary excretion rate of MG (urine MG/urine Cre) significantly increased after HBO therapy in every group. Urine MG/urine Cre/serum Cre ratio which was used as a index of MG synthesis rate also increased. In this study, it is clarified that MG excretion rate increases in hyperoxic condition. These results suggest that active oxygen plays an important role on MG synthesis in vivo, and that the urine MG/urine Cre/serum Cre ratio can be a useful maker of the active oxygen products in vivo.

Lack of detectable N-nitroso-N-methyl-N-cyclohexylamine in humans after administration of bromhexine.

The possible formation of N-nitroso-N-methyl-N-cyclohexylamine (NMCA) from the drug bromhexine (N-methyl-N-cyclohexyl-(2-amino-3,5-dibromobenzyl)-ammonium hydrochloride) and nitrite was investigated in humans using three different approaches: 1. analysis on metabolites of NMCA in human urine; 2. analysis on NMCA in human gastric juice; 3. in vitro incubation of human gastric juice with therapeutic bromhexine doses. Diet given to volunteers was varied during these investigations with respect to nitrate content. Experiments with a maximum load of 200 mg nitrate to stimulate nitrite formation were performed. Results of in vivo experiments did not indicate any formation of NMCA. In one out of 39 ex-vivo/in-vitro experiments (with a load of 100 mg nitrate in drinking water) 0.5 ng NMCA/ml gastric juice could be detected which is near the detection limit. Finally, this study showed that bromhexine is not secreted by saliva. This allows to conclude that nitrite and bromhexine do not reach the stomach simultaneously over a longer period of time. In consequence, medication with bromhexine is not regarded to represent a risk due to nitrosamine formation.

Amine metabolite profile of normal and uremic urine using gas chromatography--mass spectrometry.

A method for the simultaneous analysis of phenolic amines and aliphatic amines in human urine is described. The amine metabolites in urine were extracted using Dowex 50W-X8 cationic resin, derivatized and analyzed by a gas chromatographic--mass spectrometric--computer system. The amine metabolites profile of 5 ml of urine was obtained with good gas chromatographic separation. The gas chromatographic method described here separates urinary phenolic amines, di- and polyamines and methylguanidine in a single chromatographic separation. The urinary levels of methylguanidine, putrescine, cadaverine, spermidine, p-tyramine, dopamine, and 3-methoxytyramine were quantitated by using a mass spectrometric technique. In uremic patients, only the urinary excretion of methylguanidine was increased in comparison with normal subjects, although the urinary excretion of other amines was decreased in uremic patients.

Effect of arginine or creatinine administration on the urinary excretion of methylguanidine.

The metabolic pathway of methylguanidine (MG) is mainly speculated from the change in urinary excretion of MG in the arginine (Arg)-injected normal rat, the creatinine (Cr)-injected normal rat, and the Arg-injected uremic rat. 15N-Arg was ingested to 2 uremic patients. Arg administration resulted in marked increase in urinary MG excretion both in the uremic rat and patient, but not in the normal rat. In the first phase of the 15N-Arg ingestion experiment, a rapid rise of 15N atom percent excess of urinary MG was observed in the uremic patient. In the second phase of this study, after 24 h of 15N-Arg ingestion, the 15N atom percent excess of urinary Cr and that of MG closely paralleled. These findings imply that there might be two metabolic origins of MG: one is a formation of MG from Arg itself or an Arg metabolite other than Cr, the other a pathway producing MG via Cr. The former is compatible with the hypothesis by Cohen.

Increased urinary excretion of nucleic acid and nicotinamide derivatives by rats after treatment with alkylating agents.

Rats treated with di(2-chloroethyl)methylamine (HN2), N-methyl-N-nitrosourea (MNUA) and N-ethyl-N-nitrosourea (ENUA) excrete significantly larger amounts of deoxycytidine (dC) and thymidine in their urine 0-24 h after treatment. Ethyl methanesulphonate (EMS) and dimethylnitrosamine (DMN) gave negative results in this respect but all five alkylating agents increased the excretion of 1-methyl-nicotinamide (1-meNmd). In addition, a larger quantity of 7-methylguanine (7MG) and uric acid was excreted after DMN treatment. 1,4-Dimethanesulphonoxybutane (myleran), 2,2-dichlorovinyl dimethyl phosphate (dichlorvos), 5-fluorouracil (5FU), cytosine arabinoside (araC), 2-acetylaminofluorene (AAF) and 7-bromomethylbenz-[a]anthracene (7-BrMBA) gave negative results.

Factors affecting the metabolic production of methylguanidine.

1. Methylguanidine administered orally to normal volunteers was almost completely recovered in the urine, indicating that it is absorbed in the gastrointestinal tract and is not converted into other compounds. In normal persons at least, its urinary output therefore corresponds to its metabolic production rate plus the amount ingested. 2. In normal persons, diets based on foods not containing methylguanidine (e.g. vegetarian, protein-free and milk-egg) caused a fall in the urinary output of methylguanidine as compared with the output of the same subjects on a free diet. Conversely, higher amounts of methylguanidine were excreted on a diet rich in broth and in boiled beef, which contain large amounts of methylguanidine formed from the oxidation of creatinine, caused by boiling. 3. Oral administration of creatinine to normal volunteers induced an immediate and marked increase in urinary excretion of methylguanidine, and the ingestion of [methyl-14-C]creatinine by uraemic patients was followed by the urinary excretion of labelled methylguanidine. These findings indicate that creatinine is partly converted into methylguanidine in both normal and uraemic subjects and accounts for the high metabolic production of methylguanidine in patients with renal failure, in whom the body pool of creatinine is high. 4. Creatinine, incubated at 38 degrees C for 24 h in Krebs bicarbonate solution (pH 7-38) through which was bubbled oxygen with 15% carbon dioxide, was partially oxidized to methylguanidine. This raises the possibility that even in vivo such a conversion may occur "non-enzymatically".