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Background: Short-chain volatile amines (SCVA) are an interesting compound class playing crucial roles in physiological and toxicological human settings. Dimethylamine (DMA), trimethylamine (TMA), diethylamine (DEA), and triethylamine (TEA) were investigated in detail., Methods: Headspace gas chromatography coupled to mass spectrometry (HS-GC-MS) was used for the simultaneous qualitative and quantitative determination of four SCVA in different human body fluids. Four hundred microliters of Li-heparin plasma and urine were analyzed after liberation of volatile amines under heated conditions in an aqueous alkaline and saline environment. Target analytes were separated on a volatile amine column and detected on a Thermo DSQ II mass spectrometer scheduled in single ion monitoring mode., Results: Chromatographic separation of selected SCVA was done within 7.5 minutes. The method was developed and validated with respect to accuracy, precision, recovery and stability. Accuracy and precision criteria were below 12% for all target analytes at low and high levels. The selected extraction procedure provided recoveries of more than 92% from both matrices for TMA, DEA and TEA. The recovery of DMA from Li-heparin plasma was lower but still in the acceptable range (>75%). The newly validated method was successfully applied to plasma and urine samples from healthy volunteers. Detected concentrations of endogenous metabolites DMA and TMA are comparable to already known reference ranges., Conclusion: Herein, we describe the successful development and validation of a reliable and broadly applicable HS-GC-MS procedure for the simultaneous and quantitative determination of SCVA in human plasma and urine without relying on derivatization chemistry., (© 2019 The Authors. Journal of Clinical Laboratory Analysis published by Wiley Periodicals, Inc.)

Scope: Fish intake is reported to be associated with certain health benefits; however, accurate assessment of fish intake is still problematic. The objective of this study is to identify fish intake biomarkers and examine relationships with health parameters in a free-living population., Methods and Results: In the NutriTech study, ten participants randomized into the fish group consume increasing quantities of fish for 3 days per week for 3 weeks. Urine is analyzed by NMR spectroscopy. Trimethylamine-N-oxide (TMAO), dimethylamine, and dimethyl sulfone are identified and display significant dose-response with intake (p < 0.05). Fish consumption yields a greater increase in urinary TMAO compared to red meat. Biomarker-derived fish intake is calculated in the National Adult Nutrition Survey cross-sectional study. However, the correlation between fish intake and TMAO (r = 0.148, p < 0.01) and that between fish intake and calculated fish intake (r = 0.142, p < 0.01) are poor. In addition, TMAO shows significantly positive correlation with serum insulin and insulin resistance in males and the relationship is more pronounced for males with high dietary fat intake., Conclusion: Urinary TMAO displays a strong dose-response relationship with fish intake; however, use of TMAO alone is insufficient to determine fish intake in a free-living population., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

As a major kind of carbamate insecticide, propoxur plays an important role in agriculture, veterinary medicine, and public health. The acute toxicity of propoxur is mainly neurotoxicity due to the inhibition of cholinesterase. However, little is known regarding the toxicity of propoxur upon long-term exposure at low dose. In this study, Wistar rats were orally administrated with low dose (4.25 mg/kg body weight/day) for consecutive 90 days. And the urine samples in rats treated with propoxur for 30, 60, and 90 days were collected and analyzed by employing 1 H NMR-based metabolomics approach. We found that propoxur caused significant changes in the urine metabolites, including taurine, creatinine, citrate, succinate, dimethylamine, and trimethylamine-N-oxide. And the alteration of the metabolites was getting more difference compared with that of the control as the exposure time extending. The present study not only indicated that the changed metabolites could be used as biomarkers of propoxur-induced toxicity but also suggested that the time-course alteration of the urine metabolomic profiles could reflect the progressive development of the toxicity following propoxur exposure.

Gut microbiota-dependent metabolites trimethylamine N-oxide (TMAO), trimethylamine (TMA) and dimethylamine (DMA) from dietary methylamines have recently gained much attention due to their high association with chronic kidney disease risk. Hence a simpler and faster performance liquid chromatography-tandem mass spectrometry method was developed and validated. The quantitative analysis was achieved within 6 min by using Agilent 6460C UPLC-MS/MS with 10% methyl alcohol isocratic elution and was more simple, convenient and rapid than that of previously reported methods. Furthermore, method verification results showed that the method correlation coefficient was 0.99978293, 0.99997514 and 0.98784721, and the detection limit was 0.121, 8.063 and 0.797 μg L-1, and the precision of the retention time and peak area of analytes was less than 0.331 and 3.280, respectively. The method was applied to simultaneously determine TMAO, TMA and DMA in the urine and serum from mice treated with normal, high l-carnitine, or high choline diet. Quantitative recoveries of TMAO, TMA and DMA were in the range of 94.2%-101.0%, and the RSD values were lower than 5.17%. The proposed UPLC-MS/MS-based assay should be of value for further evaluating TMAO as a risk marker and for examining the effect of dietary factors on TMAO metabolism.

Background: Several metabolites and altered metabolic pathways have been reported to be associated with asthma. However, longitudinal analysis of the dynamics of metabolites contributing to the development of asthma has not yet been fully clarified., Methods: We sought to identify the metabolic mechanisms underlying asthma development in early childhood. Thirty children with asthma and paired healthy controls from a prospective birth cohort were enrolled. Time series analysis of urinary metabolites collected at ages 1, 2, 3, and 4 years was assessed using 1 H nuclear magnetic resonance (NMR) spectroscopy coupled with partial least squares discriminant analysis (PLS-DA). Metabolites identified were studied in relation to changes over time in a linear mixed model for repeated measures., Results: A total of 172 urine samples collected from the enrolled children were analyzed. Urinary metabolomics identified four metabolites significantly associated with childhood asthma development, with longitudinal analysis. Among them, dimethylamine, a metabolite produced by intestinal bacteria, appeared to shift from higher to lower level during asthma development. A persistent lower level of 1-methylnicotinamide and allantoin was found in children with asthma, with a peak difference at age 3 years (P = .032 and P = .021, respectively). Furthermore, a significant inverse correlation was found between allantoin and house dust mite sensitization (Spearman's r = -.297 P = .035)., Conclusions: Longitudinal urinary metabolomic profiling provides a link of microbe-environment interactions in the development of childhood asthma. 1-Methylnicotinamide and allantoin may participate in allergic reactions in response to allergen exposure, potentially serving as specific biomarkers for asthma., (© 2018 EAACI and John Wiley and Sons A/S. Published by John Wiley and Sons Ltd.)

The specific nutritional composition of nuts could affect different metabolic pathways involved in a broad range of metabolic diseases. We therefore investigated whether chronic consumption of pistachio nuts modifies the urine metabolome in prediabetic subjects. We designed a randomized crossover clinical trial in 39 prediabetic subjects. They consumed a pistachio-supplemented diet (PD, 50% carbohydrates, 33% fat, including 57 g/d of pistachios daily) and a control diet (CD, 55% carbohydrates, 30% fat) for 4 months each, separated by a 2-week wash-out. Nuclear magnetic resonance (NRM) was performed to determine changes in 24-h urine metabolites. Significant changes in urine metabolites according to the different intervention periods were found in uni- and multivariate analysis. Score plot of the first two components of the multilevel partial least squares discriminant analysis (ML-PLS-DA) showed a clear separation of the intervention periods. Three metabolites related with gut microbiota metabolism (i.e., hippurate, p-cresol sulfate and dimethylamine) were found decreased in PD compared with CD (P<.05). Moreover, cis-aconitate [intermediate of the tricarboxylic acid (TCA)] was also found decreased following PD compared with CD. Intragroup analysis showed that creatinine levels were significantly increased in PD (P=.023), whereas trimethylamine N-oxide (TMAO) was found significantly reduced following PD (P=.034). Our results suggest that chronic pistachio consumption may modulate some urinary metabolites related to gut microbiota metabolism and the TCA cycle; all associated with metabolic derangements associated with insulin resistance and Type 2 diabetes., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Understanding how metabolite levels change over the 24 hour day is of crucial importance for clinical and epidemiological studies. Additionally, the association between sleep deprivation and metabolic disorders such as diabetes and obesity requires investigation into the links between sleep and metabolism. Here, we characterise time-of-day variation and the effects of sleep deprivation on urinary metabolite profiles. Healthy male participants (n = 15) completed an in-laboratory study comprising one 24 h sleep/wake cycle prior to 24 h of continual wakefulness under highly controlled environmental conditions. Urine samples were collected over set 2-8 h intervals and analysed by (1)H NMR spectroscopy. Significant changes were observed with respect to both time of day and sleep deprivation. Of 32 identified metabolites, 7 (22%) exhibited cosine rhythmicity over at least one 24 h period; 5 exhibiting a cosine rhythm on both days. Eight metabolites significantly increased during sleep deprivation compared with sleep (taurine, formate, citrate, 3-indoxyl sulfate, carnitine, 3-hydroxyisobutyrate, TMAO and acetate) and 8 significantly decreased (dimethylamine, 4-DTA, creatinine, ascorbate, 2-hydroxyisobutyrate, allantoin, 4-DEA, 4-hydroxyphenylacetate). These data indicate that sampling time, the presence or absence of sleep and the response to sleep deprivation are highly relevant when identifying biomarkers in urinary metabolic profiling studies.

Metabonomic analysis, clinical chemical analysis and histopathology were used to investigate the toxic effects of subchronic exposure to dichlorvos, deltamethrin, and a combination of these two pesticides, in rats. Weight loss, hind limb weakness and histopathological changes in kidney tissue were only observed in rats exposed to high doses of deltamethrin, or a combination of deltamethrin and dichlorvos. Urinary metabonomic analysis indicated that exposure to a mixture of dichlorvos and deltamethrin was followed by increases in urinary lactate, dimethylamine, N-glycoprotein (NAC) and glycine similar to those observed in rats treated with either dichlorvos or deltamethrin alone. Serum metabonomic analysis suggests that dichlorvos induced an increase in lactate and alanine and a decrease in dimethylglycine (DMG), NAC and very low- and low-density lipoprotein (VLDL/LDL). High levels of lactate and low levels of NAC and VLDL/LDL were observed in the deltamethrin treatment group. Treating rats with a mixture of dichlorvos and deltamethrin caused an increase in serum lactate, trimethylamine-N-oxide (TMAO), choline and alanine, with the highest levels of these metabolites observed in those that received the highest dose. Exposure to a mixture of dichlorvos and deltamethrin also resulted in a decrease in serum acetone, DMG, NAC, and VLDL/LDL. Changes in serum TMAO, alanine, choline and acetone in this treatment group were higher than in rats treated with either dichlorvos or deltamethrin. These results suggest that exposing rats to subchronic doses of dichlorvos, deltamethrin, or a combination of these pesticides, disrupted the energy metabolism of the liver and reduced kidney function., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Cyadox is an antibiotic drug and has the potential to be used as a feedstuff additive in promoting the growth of animals. However, the toxicity of cyadox should be fully assessed before application, and this has prompted the current investigation on the metabolic responses of mice to cyadox exposure, using a metabonomic technique. Three groups of Kunming mice were respectively given a single dose of cyadox at three different concentrations (100, 650, and 4000 mg/kg body weight) via gavage. We present here the metabolic alterations of urine, plasma, liver, and renal medulla extracts induced by cyadox exposure. The metabolic alterations induced by cyadox exposure are dose-dependent, and metabolic recovery is achieved only for low and moderate levels of cyadox exposure during the experimental period. Cyadox exposure resulted in a disturbance of gut microbiota, which is manifested in depleted levels of urinary hippurate, trimethylamine-N-oxide (TMAO), dimethylamine (DMA), and trimethylamine (TMA). In addition, mice exposed to cyadox at high levels caused accumulations of amino acids and depletions of nucleotides in the liver. Furthermore, marked elevations of nucleotides and a range of organic osmolytes, such as myo-inositol, choline, and glycerophosphocholine (GPC), and decreased levels of amino acids are observed in the renal medulla of cyadox-exposed mice. These results suggest that cyadox exposure causes inhibition of amino acid metabolism in the liver and disturbance of gut microbiota community, influencing osmolytic homeostasis and nucleic acids synthesis in both the liver and the kidney. Our work provides a comprehensive view of the toxicological effects of cyadox, which is important in animal and human food safety.

Plasma concentration of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide (NO) synthesis from L-arginine and a cardiovascular risk factor, was found to be elevated in plasma of homocysteinemic adults. Enhanced cardiovascular risk due to homocystinuria and impaired renal function has been found in patients with phenylketonuria (PKU) on protein-restricted diet. However, it is still unknown whether ADMA synthesis is also elevated in children with homocystinuria due to cystathionine beta-synthase deficiency (classical homocystinuria), and whether ADMA may play a role in phenylketonuria in childhood. In the present study, we investigated the status of the L-arginine/NO pathway in six young patients with homocystinuria, in 52 young phenylketonuria patients on natural protein-restricted diet, and in age- and gender-matched healthy children serving as controls. ADMA in plasma and urine was determined by GC-MS/MS. The NO metabolites nitrate and nitrite in plasma and urine, and urinary dimethylamine (DMA), the dimethylarginine dimethylaminohydrolase (DDAH) metabolite of ADMA, were measured by GC-MS. Unlike urine ADMA excretion, plasma ADMA concentration in patients with homocystinuria was significantly higher than in controls (660±158 vs. 475±77 nM, P=0.035). DMA excretion rate was considerably higher in children with homocystinuria as compared to controls (62.2±24.5 vs. 6.5±2.9 μmol/mmol creatinine, P=0.068), indicating enhanced DDAH activity in this disease. In contrast and unexpectedly, phenylketonuria patients had significantly lower ADMA plasma concentrations compared to controls (512±136 vs. 585±125 nM, P=0.009). Phenylketonuria patients and controls had similar L-arginine/ADMA molar ratios in plasma. Urinary nitrite excretion was significantly higher in phenylketonuria as compared to healthy controls (1.7±1.7 vs. 0.7±1.2 μmol/mmol creatinine, P=0.003). Our study shows that the L-arginine/NO pathway is differently altered in children with phenylketonuria and homocystinuria. Analogous to hyperhomocysteinemic adults, elevated ADMA plasma concentrations could be a cardiovascular risk factor in children with homocystinuria. In phenylketonuria, the L-arginine/NO pathway seems not be altered. Delineation of the role of ADMA in childhood phenylketonuria and homocystinuria demands further investigation.

Introduction: Asymmetric dimethylarginine (ADMA) concentrations are increased in critically ill patients and may play a role in multiple organ failure. However, plasma ADMA concentrations during the development of the inflammatory response have not been documented. We measured plasma ADMA, as well as urinary excretion of its major metabolite dimethylamine, and nitrate as a marker of nitric oxide synthase (NOS) activity, in a cohort of patients undergoing elective knee arthroplasty that is known to provoke a significant inflammatory response., Methods: Thirty-eight patients were recruited. Fasting venous blood samples were obtained pre-operatively and at 12h and daily until the fifth post-operative day. ADMA and symmetric dimethylarginine (SDMA) were measured by high-performance liquid chromatography (HPLC). Urinary dimethylamine and nitrate were measured pre-operatively and on each of the post-operative mornings using HPLC and expressed as a ratio to creatinine., Results: Plasma ADMA fell by a median of 31% during the post-operative period, reaching a nadir on day 2, and recovering to baseline by the end of the study. SDMA showed no significant changes. No increase in urinary dimethylamine excretion was noted until day 5 post-op, whereupon it doubled. Urinary nitrate showed a small, but nonsignificant decrease on day 2, suggesting no major activation of NOS activity., Conclusions: Plasma ADMA concentration decreases rapidly and transiently during the first 48h of acute inflammation. This appears not be caused by increased catabolism and may reflect increased cellular partitioning. This may serve to regulate NOS activity and prevent harmful increases in inducible NOS in situations where it is not appropriate., (© 2010 The Authors. European Journal of Clinical Investigation © 2010 Stichting European Society for Clinical Investigation Journal Foundation.)

To further investigate pathogenesis and pathogenic process of type 2 diabetes mellitus (T2DM), we compared the urinary metabolic profiling of Zucker obese and Goto-kakizaki (GK) rats by NMR-based metabonomics. Principal component analysis (PCA) on urine samples of both models rats indicates markedly elevated levels of creatine/creatinine, dimethylamine, and acetoacetate, with concomitantly declined levels of citrate, 2-ketoglurarate, lactate, hippurate, and succinate compared with control rats, respectively. Simultaneously, compared with Zucker obese rats, the GK rats show decreased levels of trimethylamine, acetate, and choline, as well as increased levels of creatine/creatinine, acetoacetate, alanine, citrate, 2-ketoglutarate, succinate, lactate, and hippurate. This study demonstrates metabolic similarities between the two stages of T2DM, including reduced tricarboxylic acid (TCA) cycle and increased ketone bodies production. In addition, compared with Zucker obese rats, the GK rats have enhanced concentration of energy metabolites, which indicates energy metabolic changes produced in hyperglycemia stage more than in insulin resistance stage.

The L-arginine/nitric oxide (L-Arg/NO) pathway is altered in liver and kidney diseases. However, the status of the L-Arg/NO pathway during and after orthotopic transplantation is insufficiently investigated and findings are uncertain because of analytical shortcomings. Also, most human studies have focused on individual members of the L-Arg/NO pathway such as nitrate or asymmetric dimethylarginine (ADMA). In the present article we report on a pilot study investigating extensively the status of the L-Arg/NO pathway before and during orthotopic liver transplantation (OLT). By using fully validated, highly sensitive and specific GC-MS and GC-MS/MS methods nitrite, nitrate, ADMA and its hydrolysis product dimethylamine (DMA), L-arginine and L-ornithine were measured in blood and urine. Our study gives strong evidence of the exceptional importance of hepatic dimethylarginine dimethylaminohydrolase (DDAH) activity for the elimination of systemic ADMA. In end-stage liver disease the synthesis of NO and ADMA as well as the DDAH activity are elevated. However, increase in DDAH activity is insufficient to efficiently eliminate overproduced ADMA. The transplanted liver graft is capable of clearing ADMA in a rapid and sufficient manner. In contrast to studies from other groups, our study shows that in OLT as well as in living donor kidney transplantation, the second study reported here, reperfusion of the graft does not cause drastic alterations to the L-Arg/NO pathway with regard to NO synthesis. In the OLT study the concentration of circulating L-arginine fell temporally dramatically, while L-ornithine levels increased diametrically, most likely due to elevation of arginase activity. However, the relatively long-lasting decrease in plasmatic L-arginine in OLT seems not to have affected NO synthesis after reperfusion. Our OLT study suggests that liver reperfusion is associated with greatly elevated activity of proteolytic and hydrolytic enzymes including DDAH and arginase. Suppression of proteolytic and hydrolytic activity in transplantation could be a useful measure to improve outcome and remains to be investigated in further studies on larger patient collectives. The importance of analytical chemistry in this area of research is also discussed in this article.

Forty-six different foods eaten by six healthy male volunteers were investigated as potential sources of the aliphatic secondary amine, dimethylamine. None that were representatives from the fruit and vegetable, meat, dairy and grain produce categories afforded any measurable elevation in urinary dimethylamine output following ingestion. All of the statistically significant increases occurred after consumption of fish and seafoods. However, within this category a wide variation was observed. The highest values were obtained for coley, squid and whiting with cod, haddock, sardine, skate and swordfish also producing substantial increases. Freshwater trout, plaice and prawns gave no discernable effect. It seems that not all fish and seafoods may be treated equally with regards to human dimethylamine exposure and that the situation is more complicated than at first appears.

Dimethylamine [DMA, (CH(3))(2)NH)] is abundantly present in human urine. Main sources of urinary DMA have been reported to include trimethylamine N-oxide, a common food component, and asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide (NO) synthesis. ADMA is excreted in the urine in part unmetabolized and in part after hydrolysis to DMA by dimethylarginine dimethylaminohydrolase (DDAH). Here we describe a GC-MS method for the accurate and rapid quantification of DMA in human urine. The method involves use of (CD(3))(2)NH as internal standard, simultaneous derivatization with pentafluorobenzoyl chloride and extraction in toluene, and selected-ion monitoring of m/z 239 for DMA and m/z 245 for (CD(3))(2)NH in the electron ionization mode. GC-MS analysis of urine samples from 10 healthy volunteers revealed a DMA concentration of 264+/-173 microM equivalent to 10.1+/-1.64 micromol/mmol creatinine. GC-tandem MS analysis of the same urine samples revealed an ADMA concentration of 27.3+/-15.3 microM corresponding to 1.35+/-1.2 micromol/mmol creatinine. In these volunteers, a positive correlation (R=0.83919, P=0.0024) was found between urinary DMA and ADMA, with the DMA/ADMA molar ratio being 10.8+/-6.2. Elevated excretion rates of DMA (52.9+/-18.5 micromol/mmol creatinine) and ADMA (3.85+/-1.65 micromol/mmol creatinine) were found by the method in 49 patients suffering from coronary artery disease, with the DMA/ADMA molar ratio also being elevated (16.8+/-12.8). In 12 patients suffering from end-stage liver disease, excretion rates of DMA (47.8+/-19.7 micromol/mmol creatinine) and ADMA (5.6+/-1.5 micromol/mmol creatinine) were found to be elevated, with the DMA/ADMA molar ratio (9.17+/-4.2) being insignificantly lower (P=0.46). Between urinary DMA and ADMA there was a positive correlation (R=0.6655, P<0.0001) in coronary artery disease, but no correlation (R=0.27339) was found in end-stage liver disease.

The relationships between several small molecular weight aliphatic amines (methylamine, dimethylamine, trimethylamine, and ethylamine) and an associated N-oxide (trimethylamine N-oxide) quantified in human urine collected from 203 healthy volunteers have been assessed mathematically. Principal component analysis highlighted a female subgroup with raised trimethylamine levels and the possibility of hormonal influence on the N-oxidation of trimethylamine has been proposed. A second subgroup of men, who ate a large meal of fish before the study, displayed raised levels of all compounds except ethylamine. In all cases, ethylamine was least significantly correlated with the other urinary components and appeared metabolically unrelated.

Mice provide a range of important models of human disease. As part of a broad program of metabolic phenotyping (metabotyping) the effects of gender and strain upon urinary metabolite composition and variation have been investigated using 1H NMR spectroscopy and chemometrics in the Alpk:ApfCD, C57BL10J and the "Nude mouse". By using Principal Components Analysis (PCA) and Soft Independent Modeling by Class Analogy (SIMCA), characteristic metabotypes for each strain were produced for both male and female animals. In all three strains, female urinary metabolic profiles were characterized by higher lactate, trimethylamine-N-oxide and lower trimethylamine concentrations relative to males. Both male and female Nude mice were phenotypically distinct from the Alpk:ApfCD and C57BL10J strains-the Nude mouse phenotypes being characterized by higher urinary creatinine, guanadinoacetic acid, dimethylamine, alpha-hydroxy-N-valeric acid and taurine levels and lower hippurate, citrate, creatine and succinate concentrations relative to those excreted by the two phenotypically normal mouse strains. These data show that Nude mice exhibit a wide variety of metabolic differences across a much wider range of pathways than has previously been thought, with potentially important considerations for studies that use the Nude mouse as a mouse model.

Occasional or suicidal methanol intoxications are a permanent problem for most Poisoning Centers around the World. Therefore it is important to look for new diagnostic and clinical prognostic methods. In the present paper 5 cases of methanol intoxication were analyzed. At first the methanol concentrations in blood and urine were estimated with headspace gas chromatography technique. Next the urine samples were examined with 1H NMR spectroscopy, then the levels of ethanol, methanol and its metabolite, formate, lactate and trimethylamine-N-oxide with dimethylamine were evaluated. The concentrations of the above compounds were correlated with the patient's clinical status, the level of ethanol and methanol and biochemical parameters. The results indicate the correlation between clinical course of intoxication, prognostication and lactate level. There were no significant parallels for formate level as acidosis causing metabolite and initial methanol levels. In the urine samples of intoxicated patients the increased trimethyl-N-oxide and dimethylamine levels were observed, which may indicate renal cortex damage. Contrary to the opinion of some clinicians, methanol intoxication may be connected with renal functional disturbances. 1H NMR examination of urine appears to be an excellent tool to evaluate the clinical course of methanol intoxication.

Genetically hypertensive rats provide a simple and accessible model for studying essential hypertension, which is a polygenic, heterogenous and multifactorial disease. Their genetic and metabolic features are of great interest because they may provide insight into the pathophysiological processes underlying essential hypertension. We have investigated the genetic influence on metabolic balance and metabolite excretion patterns in stroke-prone spontaneously hypertensive rats (SHRSP) with established hypertension using 1H NMR-based metabonomics. Urinary metabolite profiles for SHRSP and their age-matched normotensive controls, Wistar Kyoto rats, were acquired using 1H NMR spectroscopy. Principal components analysis was applied to these complex NMR data to facilitate differentiation and determine metabolic differences between urine samples collected from the hypertensive and normotensive rats. Consequently, it was possible to distinguish urine samples between the two strains in the principal components scores plot. The loadings plot showed that taurine, creatine and some unidentified metabolites resonating at around delta 2.48, 3.10 and 3.58 predominantly contributed to the separation. In SHRSP, the urinary levels of taurine and creatine were found to be higher and the intensities of the unknown signals much lower than those in the Wistar Kyoto rats. Although the pathophysiological significance of these components remains to be elucidated, this study suggests that 1H NMR-based metabonomics is a promising approach to provide new information on metabolic changes related to the pathophysiological processes of the genetically hypertensive rats.

Injury during the transplant process affects the alloantigen-dependent factors and the alloantigen-independent processes of "chronic" rejection. Consequently, the determination of reliable parameters for the assessment of ischemic damage is essential for the prediction of renal changes after ischemia/reperfusion injury. The aim of this study was to assess the ability of (1)H NMR spectroscopy to predict the early graft dysfunction in an ischemia/reperfusion model after preservation in two standard preservation solutions, Euro-Collins (EC) and University of Wisconsin (UW). The second aim was to specify the role of the UW solution in preventing renal medullary injury. Urine and plasma samples from three experimental groups were examined during 2 weeks: control group (n = 5), EC group (cold flushed and 48-h cold storage of kidney in EC and autotransplantation, n = 12), and UW group (cold flushed and 48-h cold storage of kidney in UW and autotransplantation; n = 12). We also examined these kidneys 30-40 min after implantation and on the sacrifice day. Creatinine clearance was significantly reduced in the EC group during the second week. Fractional excretion of sodium and urine N-acetyl-beta-d-glucosaminidase activity were improved but not significantly different in the preserved groups. Urinary concentrations of the alpha-class glutathione S-transferase were significantly greater in the EC group during the first week after transplantation. The most relevant resonances for evaluating renal function after transplantation determined by (1)H NMR spectroscopy were those arising from citrate, dimethylamine (DMA), lactate, and acetate in urine and trimethylamine-N-oxide (TMAO) in urine and plasma. These findings suggest that graft dysfunction is associated with damage to the renal medulla determined by TMAO release in urine and plasma associated with DMA and acetate excretion. Citrate is also a urinary marker that can discriminate kidneys with a favorable evolution. Our results suggest that (1)H NMR spectroscopy is an efficient technique for detecting ischemic damage when accurate and precise data on graft injury is required. In addition, this study outlines the specific impact of the UW solution against injury to the renal medulla., (Copyright 2000 Academic Press.)

Ischemia/reperfusion injury leads to delayed graft function, which is a major problem in kidney transplantation. This study investigated the effects of adding trimetazidine (TMZ) to the perfusate of cold-stored kidneys on the function of reperfused autotransplanted pig kidney. The left kidney was removed and cold-flushed with Euro-Collins (EC), or University of Wisconsin (UW) solutions with or without 10(-6)M TMZ and stored for 48 h at 4 degrees C. The kidneys were then autotransplanted and the contralateral kidneys were removed. Several parameters were analyzed over the 14 d after transplantation. The survival rate was 57% in pigs transplanted with kidneys cold-flushed with UW and 43% for those flushed with EC solution; it was 100% for pigs having kidneys cold-flushed with TMZ-supplemented UW and EC solutions. The functions of the transplanted kidneys were also better preserved after cold flush with TMZ-supplemented solutions than with TMZ-free solutions. Creatinine clearance was higher and the urinary excretion of trimethylamine-N-oxide and dimethylamine, used as markers of renal medulla injury, were lower in animals transplanted with kidneys cold-flushed with TMZ-supplemented solutions than with TMZ-free solutions. The cytoprotective action of TMZ also reduced interstitial and peritubular inflammation and the numbers of infiltrating mononuclear CD45+and CD3+ T cells. These results indicate that the tissue damage due to ischemia/reperfusion injury may be prevented, at least in part, by adding TMZ to preservation solutions.

Dimethylamine is the immediate precursor of dimethylnitrosamine, a known potent carcinogen in a wide variety of animal species. Although small amounts of dimethylamine are ingested directly, the major dietary source is believed to be via choline and related materials. Owing to quantitative recoveries following oral administration, urinary dimethylamine levels provide good overall measures of body exposure. The oral administration of equimolar amounts (1 mmol/kg body weight) of potential amine precursors to male Wistar rats produced only small increases in urinary dimethylamine after choline (+ 11%; 0.60 +/- 0.36% dose), dimethylaminopropanol (+ 32%; 1.49 +/- 0.30% dose), dimethylaminoethyl chloride (+ 110% 5.38 +/- 1.72% dose) and trimethylamine (+ 51%; 1.6 +/- 0.80% dose) input, whereas significantly larger increases were found following trimethylamine N-oxide ingestion (+ 355%; 12.93 +/- 1.13% dose; t-test, P < 0.001). These data suggest that trimethylamine N-oxide is a major dietary source of dimethylamine, by direct conversion and not by sequential reduction (to trimethylamine) and demethylation, and that in this respect it is of greater importance, on a molar basis, than choline.

Urines from 25 normal subjects living in Rome and 25 normal subjects living in Ny-Alesund (Svaldbard) were analysed by 1HNMR spectroscopy. The observed differences in the concentration of the major metabolites were correlated to the composition of the diet. It was found that a diet rich of carbohydrates, such as the Italian diet, is responsible for an increased excretion of citrate, lactate, alanine, and glycine. Thus, a correct diagnostical interpretation of urinary metabolites needs to consider feeding habits.

A reversed-phase HPLC method for the quantification of dimethylamine in serum and urine is presented. Dimethylamine (DMA) is converted into a stable fluorescent product by precolumn derivatization with fluorenylmethylchloroformate. The DMA derivative is resolved from derivatives of other amines and amino acids by gradient elution with a total run-time of 15 min. The lower limit of determination in biological samples is 0.1 micromol/l. Recoveries from spiked serum samples were 99-107%. Within- and between-run precision were better than 6%. Concentrations of DMA in serum from normal human subjects (n=8) and from continuous ambulatory peritoneal dialysis patients (n=15) were 3.3+/-1.5 and 29.1+/-12.1 micromol/l, respectively.

The urinary excretion of dimethylamine has been measured in 203 unrelated healthy volunteers (102 male) who maintained their normal diets. The results for female volunteers are the first reported in the literature. The average daily output was 17.43 +/- 11.80 mg (mean +/- S.D.) (21.21 +/- 14.78 male; 13.74 +/- 5.65 female) with values for the majority of the population lying within the 0.68-35.72 mg range. Four male outliers excreted up to 109.2 mg; these large amounts of dimethylamine were presumed to be of dietary origin. The literature pertaining to urinary levels of dimethylamine has been summarised and integrated with the present observations.

1. [U-14C]-dimethylamine hydrochloride was administered by gavage (20 mumol/kg body weight) to adult male Wistar rat and CD1 strain mouse. 2. In both species, urine was the main route of excretion with the majority of radiolabel (91%) being voided during the first day. Additional small amounts of radioactivity were detected in the 24-72 h urine (2%), in faeces (2%) and amidst exhaled air (1%), with minor amounts remaining within the carcass (1%) after 3 days. 3. Metabolism was limited to demethylation, with the majority of the compound (89% dose; 96% urinary radioactivity) being excreted unchanged.

Fish contain methylamines, especially trimethylamine N-oxide (TMAO), trimethylamine (TMA), and dimethylamine (DMA). Further, DMA may be formed TMA and TMAO. DMA is a precursor of nitrosodimethylamine (NDMA), which is a potent carcinogen. Levels of DMA, TMA, and TMAO in urine were used as indicators of the dietary exposure and in vivo formation of these amines in 44 men, representing 3 groups with different fish consumption habits. The levels of TMA (median 0.24 mmol/mol creatinine; range 0-2.7) and TMAO (median 38 mmol/mol creatinine; range 8-290) were significantly associated with the weekly intake of fish (r = .47, p = .001, and r = .53, p = .0002, respectively), while no such relation was found for DMA (median 24 mmol/mol creatinine; range 5-46). Further, urinary levels of TMA and TMAO were dependent on recent intake of fish.

Experiments were performed with rats to examine the sources and disposition of dimethylamine (DMA), trimethylamine (TMA), and trimethylamine N-oxide (TMAO), all potential substrates for in vivo nitrosation to form N-nitrosodimethylamine (NDMA), a potent carcinogen. When bolus doses of [14C]DMA or [14C]TMA were given ip, recovery of radioactivity in the urine was essentially complete, and respiratory excretion, fecal excretion, and accumulation in tissues of these amines or their metabolites were negligible. Urine analysis following doses of stable isotopes showed that DMA was not converted to TMA or TMAO. Varying amounts of TMA were oxidized to TMAO, the fraction oxidized decreasing at higher doses of TMA. Ingestion and excretion of naturally occurring methylamines were monitored over a 5-day period in separate groups of normal and germ-free rats. The results of these metabolic balance studies indicate that there is net synthesis of DMA by gut bacteria and net consumption of TMAO by endogenous processes. The net intake or excretion of TMA and TMAO observed in normal and germ-free rats is consistent with bacterial synthesis of TMA followed by its almost complete oxidation to TMAO. Blood concentrations of DMA and TMA were measured in rats for 8 hr following < 5, 100, or 1000 mumol bolus i.v. or ip doses of radioisotopes or stable isotopes. At any given dose of DMA or TMA, the decay in blood concentration was approximately monoexponential. At the lowest (most physiologic) dose the apparent volume of distribution (VD) for DMA was larger than that for TMA. Both values of VD greatly exceeded the size of the animals, suggesting that DMA and TMA are highly concentrated at one or more locations in the body. This was confirmed by measurements in tissue homogenates sampled 1 hr after a dose. The overall handling of methylamines by rats is generally consistent with observations in humans. The presence of high local concentrations of DMA and TMA at various extragastric sites merits further investigation in connection with the potential for endogenous nitrosation of these methylamines to form NDMA.

Trimethylamine N-oxide, a common food component, has been identified as a major source of urinary dimethylamine in man. The potential pathophysiological consequences of exposure to dietary derived dimethylamine are raised.

A selective and sensitive method has been developed for the determination of secondary amines by gas chromatography (GC). After removal of primary amines by the reaction with o-phthaldialdehyde, secondary amines were converted into their N-diethylthiophosphoryl derivatives and then measured by GC with flame photometric detection using a DB-1701 capillary column. The derivatives were sufficiently volatile and stable to give single symmetrical peaks. The detection limits of secondary amines were ca. 0.05-0.2 pmol per injection. N-Methylcyclohexylamine was used as an internal standard. The calibration curves for secondary amines in the range 1-20 nmol were linear and sufficiently reproducible for quantitative determination. This method was successfully applied to small urine samples without prior clean-up. Overall recoveries of secondary amines added to urine samples were 91-105%. By using this method, secondary amines in urine samples could be analysed without any influence from primary amines and other coexisting substances. The analytical results of secondary amine content in urine samples of normal subjects are presented.

High field 1H NMR spectroscopy was used for the rapid multicomponent analysis of low molecular wt compounds in urine in order to investigate the patterns of metabolic changes associated with early renal allograft dysfunction. Urine samples were collected daily for 14 days from 33 patients who underwent primary renal allograft transplantation, and analyzed by 500 and/or 600 MHz 1H NMR spectroscopy. All patients received 20 mg prednisolone and 5 mg/kg b.d. oral cyclosporin A (CsA) solution. In this study no patient showed clinical or histopathological evidence of CsA nephrotoxicity. For each patient the NMR-generated metabolite data were correlated with the clinical observations, graft biopsy pathology, and data from conventional laboratory techniques for assessing renal function. The NMR spectra of urine from patients with immediate functioning grafts were similar with respect to their patterns of amino acids, organic acids and organic amines, whereas the patients with delayed or non-functioning grafts showed significantly different metabolite excretion patterns. In longitudinal studies on individual patients there were increased urinary levels of trimethylamine-N-oxide (TMAO), dimethylamine (DMA), lactate, acetate, succinate, glycine and alanine during episodes of graft dysfunction. However, only the urinary concentration of TMAO was statistically significantly higher (P < 0.025) in the urine collected from patients during episodes of graft dysfunction (410 +/- 102 microM TMAO/mM creatinine) than in patients with good graft function (91 +/- 18 microM TMAO/mM creatinine) or healthy control subjects (100 +/- 50 microM TMAO/mM creatinine). These findings suggest that graft dysfunction is associated with damage to the renal medulla which causes the release of TMAO into the urine from the damaged renal medullary cells. This provides a possible novel urinary marker for post-transplant graft dysfunction. This study shows that NMR spectroscopy of biofluids, when used in combination with conventional laboratory techniques, is a valuable aid to renal transplant monitoring.

Percutaneous absorption of the 14C-ring-labeled phenoxy herbicide 2,4-D-amine (2,4-dichlorophenoxyacetic acid dimethylamine) was examined following topical applications of the herbicide to the palm and forearm of human volunteers. The effect of two vehicles (water and acetone) and the mosquito repellent DEET (N,N-diethyl-m-toluamide) on dermal absorption of 2,4-D-amine also was investigated. The total percent dermal absorption was calculated from the mean percent urinary recoveries and was not corrected for nonurinary excretion. The data revealed 14 +/- 4.5% (standard deviation) and 10 +/- 11.5% palmar absorption of 2,4-D-amine applied in water, with and without DEET, respectively, and 7 +/- 6.2% and 13 +/- 5.0% forearm absorption of the herbicide applied in water or acetone, respectively. Soap-and-water skin washes conducted at 24 h posttreatment removed up to 34% of the applied dose. Successive tape strips of skin taken at 24 h posttreatment demonstrated generally decreasing herbicide levels in the outer layers. The data bring into question the complete validity of the rhesus monkey model to predict human dermal absorption.

The percutaneous penetration of 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4-D dimethylamine salt (DMA) was evaluated separately in five male volunteers who participated in both experiments. Urine samples collected for 144 h following dermal applications of 10 mg to the dorsum of the hand (9 cm2) were analyzed for 2,4-D. Following the acid application, an average of 4.46 +/- 0.849% was recovered in the urine and a significantly lower amount of 1.76 +/- 0.568% following the DMA application. Significantly higher amounts of 2,4-D DMA (7.68 +/- 0.493 mg) were washed off the hand 6 h following application as compared with 2,4-D acid (5.35 +/- 0.384 mg). These results indicate that, in addition to the differences in physical and chemical properties of the two compounds that will affect absorption, the amount of the chemical absorbed is related inversely to the amount of washed off. Urinary excretion of 2,4-D was not complete in all volunteers 144 h following either application, but in all cases it was approaching the limit of detection. An average of 84.8 +/- 2.55% and 76.8 +/- 8.05% of the total recovered in 144 h was recovered in the urine 96 h following 2,4-D acid and 2,4-D DMA application, respectively. Average, approximated half-lives for excretion were 39.5 +/- 8.1 h for the acid application and 58.5 +/- 13.2 h for the DMA application.

We have used 1H-, 13C- and 14N-NMR spectroscopy to investigate the constituents of plasma and urine in 16 patients with chronic renal failure (CRF). Resonances not previously observed in spectra of plasma from healthy volunteers were seen in CRF plasma, including those for trimethylamine-N-oxide (TMAO) and dimethylamine (DMA). A possible analogy with the plasma of elasmobranch fishes, in which TMAO stabilizes proteins in the presence of very high urea concentrations, is noted. The intensity of the TMAO resonance for CRF subjects was correlated with the plasma concentration of urea (R = 0.55) and creatinine (R = 0.74), suggesting that the presence of TMAO is closely related to the degree of renal failure. When normal subjects ate a meal of TMAO-containing fish, TMAO appeared rapidly in the plasma and in the urine. Thus TMAO is efficiently cleared by the healthy kidney. Differences in the interaction of lactate with plasma proteins were detected by NMR, suggesting that uraemia impairs their transport roles.

Various methods of preparing dermal application sites in Fischer 344 rats prior to exposure to 2,4-dichlorophenoxyacetic acid dimethylamine salt (2,4-D amine) and the effect of various cleansing agents following exposure were examined by measuring recoveries of 14C-labeled 2,4-D amine in skin, postapplication cleansing solution, blood, and urine. The middorsal area of the rat was the site of application for four treatments tested: (1) hair clipping only, (2) hair clipping followed by an epilatory cream, (3) hair clipping plus shaving with an electrical razor, and (4) as in treatment 3 followed by washing with soap and water. A last preparation was the rat's tail thoroughly brushed with soap and water. The results indicated that the tail retained greater than 75% of the material, thus preventing its absorption into the blood stream and subsequent removal by cleansing. With treatment 1 the dense short hair remaining after clipping impaired the absorption of 2,4-D as evidenced by considerably lower blood and urinary levels than treatments 2-4. With preparations 1-4, 45-61% of the dose was removed with the 7-h postapplication cleansing and a further 5-6% with the subsequent 23-h cleansing. In other studies using preparation 3 above, the following cleansing agents were tested: soap and water, water, isopropanol, acetone, and Rad-Con, a foam-producing cleanser. Rad-Con removed more 2,4-D from the skin than other cleansing agents after 7 h of exposure and more than soap and water after 23 h. The percentages of 2,4-D left on the skin following either 7- or 23-h cleansing with Rad-Con were 8-12%, nearly half those following the other cleansing agents. Cleansing agents other than Rad-Con presented little advantage over soap and water. With all cleansing agents, delaying cleansing from 7 to 23 h after exposure resulted in higher blood and urinary levels of 2,4-D measured 24 h after application.

Humans ingest substantial amounts of choline and lecithin as part of common foods. Physicians have recently begun administering large doses of these compounds to individuals with neurological diseases. A significant fraction of ingested choline is destroyed by enzymes within gut bacteria, forming trimethylamine (TMA), dimethylamine (DMA) and monomethylamine (MMA). Some of these methylamines are eventually excreted into the urine, presumably after being absorbed and carried to the kidneys via the bloodstream. The methylamines formed after choline is eaten could be substrates for the formation of nitrosamines, which have marked carcinogenic activity. Twenty-seven millimoles of choline chloride, choline stearate or lecithin were administered to healthy human subjects. It was found that these treatments markedly increased the urinary excretion of TMA, DMA and MMA, with choline chloride having the greatest effect. Rats were treated with 2 mmol/kg b.wt. of choline chloride or lecithin, and it was found that these treatments significantly increased urinary TMA excretion and did not alter DMA or MMA excretion. Our choline chloride preparation contained no MMA, DMA or TMA; however, it was found that our choline stearate and all the commercially available lecithins tested were contaminated with methylamines. Prior removal of methylamines from our lecithin preparation minimized the effect of oral administration of this compound on methylamine excretion in urine of rats and humans.

In the gastro-intestinal tract of male Wistar rats fed a commercial diet containing 23.6 ppm dimethylamine (DMA), the concentration of DMA was highest (11.2 +/- 2.1 ppm) in the stomach and declined towards the lower regions. In contrast, the highest DMA concentration (6.6 +/- 2.5 ppm) was observed in the upper small intestine in rats fed a diet containing only 1.0 ppm DMA. DMA was absorbed in the intestines, and the disappearance curves were monoexponential. The t1/2 values for DMA in the ligated stomach, upper and lower small intestine, caecum and large intestine were 198, 8.3, 11.6, 31.5 and 11.0 min, respectively. The DMA concentration in the blood had increased to 3.0 +/- 1.0 ppm (from a pre-injection level of 0.28 +/- 0.06 ppm) 5 min after the injection of 250 micrograms DMA into the ligated upper small intestine. The disappearance curve for DMA in the blood was monoexponential and the t1/2 for the initial 15 min was 12.5 min when 250 micrograms DMA was injected into a femoral vein. The peak concentrations of DMA in the intestine and bile, respectively, were 15.6 +/- 12.6 ppm (at 15 min) and 3.7 +/- 1.9 ppm (at 30 min after the iv injection of DMA). In this 30-min period, urinary DMA increased from 17.3 +/- 9.4 to 139 +/- 23 ppm. These results show that, following ingestion, DMA is absorbed from the intestine into the blood, from which it disappears rapidly, the major part being excreted in the urine while a small proportion is excreted in the bile or secreted into the intestine, where it may be reabsorbed.

The metabolism of orally administered trimethylamine has been studied in 4 male volunteers at 2 dose levels. N-Oxidation was the major route of metabolism whilst N-demethylation was negligible and only significant at the higher dose level.

The excretion of aliphatic amines, methylamine, dimethylamine and trimethylamine in the urine and faeces of rats fed on a control diet and diets supplemented with creatinine, lecithin or choline were measured over a 14 d feeding period. The rats were then killed and concentrations of amines in small and large intestinal contents measured. Adding creatinine to the diet resulted in a significant increase of methylamine excretion in the faeces and urine. The amount of methylamine found in all parts of the intestine increased, especially in the caecum. Adding lecithin to the diet resulted in an increase in the methylamine excretion only, and no change in the concentrations of amines found in the intestine, except for trimethylamine which was significantly increased in the caecum and colon. Adding choline to the diet resulted in a significant increase in excretion of trimethylamine and, to a lesser extent, methylamine. The levels of amines found in the gut increased, dimethylamine being increased in the small bowel, and methylamine and trimethylamine in the caecum.

The dimethylamine (DMA) concentration in the gastrointestinal tract of Wistar male rats fed a commercial diet containing 23.6 mg/kg DMA was highest (11.2 +/- 2.1 mg/kg) in the stomach and decreased from the upper region to the lower region. In contrast, the highest DMA concentration (6.6 +/- 2.5 mg/kg) in the upper small intestine was observed in rats fed a low-DMA diet containing 1.0 mg/kg DMA. DMA absorption was observed in the intestines and the absorbtion curves were monoexponential. The biological half-lives (t1/2) of DMA in the ligated stomach, upper and lower small intestine, caecum and large intestine were 198, 8.3, 11.6, 31.5 and 11.0 min, respectively. The DMA concentration in blood increased from 0.3 +/- 0.1 mg/kg to 3.0 +/- 1.0 mg/kg 5 min after injection of 250 micrograms DMA into the ligated upper small intestine. The disappearance curve of DMA in blood was monoexponential and the half-life for the initial 15 min was 12.5 min when 250 micrograms DMA were injected through a femoral vein. Intestinal secretion of DMA (15.6 +/- 12.6 mg/kg) was observed 15 min after the injection. Urinary DMA increased from 17.3 +/- 9.4 to 139 +/- 23 mg/kg within 30 min of intravenous injection of DMA. These results show that the behaviour of DMA in rats is as follows: (Formula: see text)

A simple gas chromatographic method for the measurement of low molecular weight urinary monoamines has been developed without use of a prior isolation procedure. The primary and secondary monamines in urine were directly converted into their 2,4-dinitrophenyl derivatives with 2,4-dinitrobenzensulfonate in aqueous alkaline solution, which after extraction with benzene and concentration, were submitted to gas chromatography. The application of this method to the determination of methylamine and dimethylamine in urine from normal subjects and hepatic patients is described.