Dietary protein and fatty acid composition of liver lipids in the rat

In order to compare the effects of different sources of dietary protein on the fatty acid composition of phosphatidylcholines (PC), phosphatidylethanolamines (PE), phosphatidylinositols (PI), cholesteryl esters and triacylglycerols, male rats were fed for a 4-week period on cholesterol-free, or cholesterol-containing, diets based on casein, or soybean protein and olive oil. The most conspicuous difference observed was the occurrence of significantly higher levels of 5,8,11-eicosatrienoic acid, 20:3 (n - 9), in the different lipid classes of casein-fed, compared with soybean protein-fed, animals. In the PI fraction of livers from the groups of rats fed casein diet, this fatty acid amounted to between 9.9 and 13.3% by weight of the total fatty acids. Phospholipids from livers of casein-fed rats contained increased levels of oleic acid, 18:1 (n - 9) (in PC and PE) and reduced levels of stearic acid (18:0). Moreover, in this group of rats PI contained a reduced level of arachidonic acid, 20:4 (n - 6). A casein-related decrease in the linoleic acid, 18:2 (n - 6), content of PC and PE was observed only in the rats fed on cholesterol-free diet. Effects on the fatty acid composition were also observed in the triacyglycerol and cholesteryl ester fractions, in which the rats fed casein diet showed higher levels of palmitoleic acid, 16:1 (n - 7) (cholesterol-supplemented diet) and lower values for linoleic acid, than the soybean protein-fed rats.     

Alterations in urinary metabolites due to unilateral ureteral obstruction in a rodent model

Urinary tract obstruction (UTO) results in renal compensatory mechanisms and may progress to irrecoverable functional loss and histologic alterations. The pathophysiology of this progression is poorly understood. We identified urinary metabolite alterations in a rodent model of partial and complete UTO using (1)H nuclear magnetic resonance ((1)H-NMR) spectroscopy. Principal component analysis (PCA) was used for classification and discovery of differentiating metabolites. UTO was associated with elevated urinary levels of alanine, succinate, dimethylglycine (DMG), creatinine, taurine, choline-like compounds, hippurate, and lactate. Decreased urinary levels of 2-oxoglutarate and citrate were noted. The patterns of alteration in partial and complete UTO were similar except that an absence of elevated urinary osmolytes (DMG and hippurate) was noted in complete UTO. This pattern of metabolite alteration indicates impaired oxidative metabolism of the mitochondria in renal proximal tubules and production of renal protective osmolytes by the medulla. Decreased production of osmolytes in complete obstruction better elucidates the pathophysiology of progression from renal compensatory mechanisms to irrecoverable changes. Further confirmation of these potential biomarkers in children with UTO is necessary.     

Analysis of plasma hippurate in humans using gas chromatography-mass spectrometry: concentration and incorporation of infused [15N]glycine

To allow in vivo determination of synthetic rates for individual proteins, physiological incorporation of infused [15N]glycine into urinary hippuric acid has been used as an indicator of intrahepatic tracer dilution. Although the kidneys might contribute to hippurate production, the relationship between hepatic, plasma, and urinary hippurate has not yet been established in humans. To further investigate these issues we developed a fast, sensitive, and reliable method for measuring simultaneously hippurate concentrations and in vivo tracer incorporation into hippurate in plasma and urine using stable isotopes and gas chromatography-mass spectrometry. We then tested this assay under several experimental conditions. Reference compounds [( 15N]- and [ring-2H5]hippurate) were synthesized and gave linear standard curves. Postabsorptive hippurate plasma levels in healthy subjects ranged from 1.2 to 10.5 microM and protein binding was 79 +/- 6% (mean +/- SD). Following a bolus dose of [15N]glycine tracer appeared in plasma hippurate; enrichment in hippurate was indistinguishable from that in glycine after an equilibration period of 20 min, indicating a close relationship between intracellular glycine and plasma hippurate. A 16-h infusion of [15N]glycine resulted in identical enrichment levels in urinary and plasma hippurate; glycine enrichment in a hepatic export protein (VLDL-ApoB) was approaching plasma hippurate but not plasma free glycine enrichment. The ability to monitor plasma hippurate is of practical advantage compared to the sampling of urine. Furthermore it allows the monitoring of rapid events in the intrahepatic dilution of an infused glycine tracer. This assay may, therefore, become an important tool in the study of hepatic protein metabolism.     

Global metabolic phenotyping in an experimental laparotomy model of surgical trauma

Surgical trauma initiates a complex series of metabolic host responses designed to maintain homeostasis and ensure survival. (1)H NMR spectroscopy was applied to intraoperative urine and plasma samples as part of a strategy to analyze the metabolic response of Wistar rats to a laparotomy model. Spectral data were analyzed by multivariate statistical analysis. Principal component analysis (PCA) confirmed that surgical injury is responsible for the majority of the metabolic variability demonstrated between animals (R2 Urine = 81.2% R2 plasma = 80%). Further statistical analysis by orthogonal projection to latent structure discriminant analysis (OPLS-DA) allowed the identification of novel urinary metabolic markers of surgical trauma. Urinary levels of taurine, glucose, urea, creatine, allantoin, and trimethylamine-N-oxide (TMAO) were significantly increased after surgery whereas citrate and 2-oxoglutarate (2-OG) negatively correlated with the intraoperative state as did plasma levels of betaine and tyrosine. Plasma levels of lipoproteins such as VLDL and LDL also rose with the duration of surgery. Moreover, the microbial cometabolites 3-hydroxyphenylpropionate, phenylacetylglycine, and hippurate correlated with the surgical insult, indicating that the gut microbiota are highly sensitive to the global homeostatic state of the host. Metabonomic profiling provides a global overview of surgical trauma that has the potential to provide novel biomarkers for personalized surgical optimization and outcome prediction.     

Variation in antibiotic-induced microbial recolonization impacts on the host metabolic phenotypes of rats

The interaction between the gut microbiota and their mammalian host is known to have far-reaching consequences with respect to metabolism and health. We investigated the effects of eight days of oral antibiotic exposure (penicillin and streptomycin sulfate) on gut microbial composition and host metabolic phenotype in male Han-Wistar rats (n = 6) compared to matched controls. Early recolonization was assessed in a third group exposed to antibiotics for four days followed by four days recovery (n = 6). Fluorescence in situ hybridization analysis of the intestinal contents collected at eight days showed a significant reduction in all bacterial groups measured (control, 10(10.7) cells/g feces; antibiotic-treated, 10(8.4)). Bacterial suppression reduced the excretion of mammalian-microbial urinary cometabolites including hippurate, phenylpropionic acid, phenylacetylglycine and indoxyl-sulfate whereas taurine, glycine, citrate, 2-oxoglutarate, and fumarate excretion was elevated. While total bacterial counts remained notably lower in the recolonized animals (10(9.1) cells/g faeces) compared to the controls, two cage-dependent subgroups emerged with Lactobacillus/Enterococcus probe counts dominant in one subgroup. This dichotomous profile manifested in the metabolic phenotypes with subgroup differences in tricarboxylic acid cycle metabolites and indoxyl-sulfate excretion. Fecal short chain fatty acids were diminished in all treated animals. Antibiotic treatment induced a profound effect on the microbiome structure, which was reflected in the metabotype. Moreover, the recolonization process was sensitive to the microenvironment, which may impact on understanding downstream consequences of antibiotic consumption in human populations.     

Mytilus trossulus hsp70 as a biomarker for arsenic exposure in the marine environment: Laboratory and real-world results

Acute renal papillary necrosis (RPN) in animals is characterized by increased renal lipid accumulation. The excretion of renal lipids into urine has been determined to evaluate their possible use as sensitive early biomarkers for the diagnosis of RPN. This study investigates injury induced by two model nephrotoxins, mefenamic acid (MFA), a non-steroidal anti-inflammatory drug (NSAID), and its analogue N-phenylanthranilic acid (NPAA). Oral NPAA was given repeatedly at doses of 100, 250 and 500 mg kg^-1 daily for 5 days, followed by a 2 day respite over the weekend, and then four further daily doses. The same dosing procedure was used with MFA, but at doses of 75, 150 and 300 mgkg^-1. The control groups were given vehicle orally using the same volume given to the test groups. Urinary phospholipids (PLs), notably sphingomyelin (SPM), phosphatidylcholine (PC) and phosphatidylethanolamine (PE), were measured and compared with other urinary parameters. Histopathological investigations were also performed to confirm the presence or absence of RPN. Following MFA treatment, PC, PI and PE were raised significantly (p < 0.001) on days 1 and 3 and for the remaining part of the experiment. After NPAA treatment, PI showed a transient elevation, and PC and PE levels were significantly increased from day 2 onwards. Both drugs caused a dose-related increase in PLs. There was no significant increase in the level of other urinary parameters. However, histopathological examination of the kidney on day 11 revealed lesions in the medulla and papilla following treatment with the two papillotoxins. These findings demonstrate the potential of urinary PLs as diagnostic non-invasive biomarkers for early renal injury associated with RPN, which may provide an important improvement in the approach to the therapeutic management of analgesic nephropathy.     

Distinct urinary metabolic profile of human colorectal cancer

A full spectrum of metabolic aberrations that are directly linked to colorectal cancer (CRC) at early curable stages is critical for developing and deploying molecular diagnostic and therapeutic approaches that will significantly improve patient survival. We have recently reported a urinary metabonomic profiling study on CRC subjects (n = 60) and health controls (n = 63), in which a panel of urinary metabolite markers was identified. Here, we report a second urinary metabonomic study on a larger cohort of CRC (n = 101) and healthy subjects (n = 103), using gas chromatography time-of-flight mass spectrometry and ultra performance liquid chromatography quadrupole time-of-flight mass spectrometry. Consistent with our previous findings, we observed a number of dysregulated metabolic pathways, such as glycolysis, TCA cycle, urea cycle, pyrimidine metabolism, tryptophan metabolism, polyamine metabolism, as well as gut microbial-host co-metabolism in CRC subjects. Our findings confirm distinct urinary metabolic footprints of CRC patients characterized by altered levels of metabolites derived from gut microbial-host co-metabolism. A panel of metabolite markers composed of citrate, hippurate, p-cresol, 2-aminobutyrate, myristate, putrescine, and kynurenate was selected, which was able to discriminate CRC subjects from their healthy counterparts. A receiver operating characteristic curve (ROC) analysis of these markers resulted in an area under the receiver operating characteristic curve (AUC) of 0.993 and 0.998 for the training set and the testing set, respectively. These potential metabolite markers provide a novel and promising molecular diagnostic approach for the early detection of CRC.     

Revealing the metabonomic variation of EC using 1H-NMR spectroscopy and its association with the clinicopathological characteristics

In this study, (1)H NMR-based metabonomics has been applied to investigate esophageal cancer metabolic signatures in plasma and urine, purpose of assessing the diagnostic potential of this approach and gaining novel insights into esophageal cancer metabolism and systemic effects. Plasma and urine samples from esophageal cancer patients (n = 108) and a control healthy group (n = 40) were analyzed by Nuclear Magnetic Resonance (NMR) spectroscopy (600 MHz), and their spectral profiles subjected to Orthogonal Projections to Latent Structures (OPLS-DA) for multivariate statistics. Potential metabolic biomarkers were identified using data base comparisons used for examining the significance of metabolites. Compared to healthy controls, esophageal cancer plasma had higher levels of dimethylamine, α-glucose, β-glucose, citric acid, together with lower levels of Leucine, alanine, isoleucine, valine, glycoprotein, lactate, acetone, acetate, choline, isobutyrate, unsaturated lipid, VLDL, LDL, 1-methylhistidine; Compared to healthy controls, esophageal cancer urine had higher levels of Mannitol, glutamate, γ-propalanine, phenylalanine, acetate, allantoin, pyruvate, tyrosine, β-glucose and guinolinate, together with lower levels of N-acetylcysteine, valine, dihydrothymine, hippurate, methylguanidine, 1-methylnicotin- amide and Citric acid; Very good discrimination between cancer and control groups was achieved by multivariate modeling of plasma and urinary profiles. (1)H NMR-based metabolite profiling analysis was shown to be an effective approach to differentiating between patients with EC and healthy subjects. Good sensitivity and selectivity were shown by using the metabolite markers discovered to predict the classification of samples from the healthy control group and the patients with the disease. Plasma and urine metabolic profiling may have potential for early diagnosis of EC and may enhance our understanding of its mechanisms.     

Metabolomic studies on the biochemical profile of urine from rats with acute cysteamine supplementation

This study investigates the effect of acute cysteamine (CS) supplementation on rat metabolism. A metabolomic strategy using high-resolution ¹H-NMR spectroscopy in conjunction with principal component analysis was applied to examine rat biological responses to CS administration. Half of female Sprague–Dawley rats (2 groups of 6 rats) were each given doses of 150 mg CS/kg body weight intraperitoneally. Urine samples were collected twice daily (0–8 and 8–24 h) from the rats following CS administration. The identifiable biochemical effects associated with CS supplementation included decreased urinary concentrations of hippurate, succinate, citric acid, and 2-oxoglutarate, as well as increased urinary concentrations of dimethylamine, dimethylglycine, glycine, and taurine. These effects were predominately seen within the first 8 h after CS administration. Clear differences in succinate, citric acid, and 2-oxoglutarate were observed 8–24 h following CS. The results suggest that CS supplementation in the rats resulted in modulation of intestinal microbial metabolism and metabolic perturbation of the tricarboxylic acid cycle.     

Decrease of hepatic delta-aminolevulinate dehydratase activity in an animal model of fatigue

Fatigue can be defined physiologically as inability to maintain the expected power output. At present, no standard of fatigue are yet available. In order to find biomarkers of fatigue, we investigated the level of delta-aminolevulinic acid (ALA), the first intermediate metabolite in the heme biosynthetic pathway, in the plasma and urine of an animal model of fatigue. To prepare fatigued animals, we kept rats for 5 days in a cage filled with water to a height of 1.5 cm. As a result, the plasma and urinary ALA levels were increased in the fatigued animals as compared with those in the control animals. One day after the rats had been returned to their normal cages, these increased levels were restored to the control ones. We also examined the activity of the enzyme ALA dehydratase (ALAD), which is the second enzyme in the heme biosynthetic pathway, and ALAD gene expression during the fatigue and its recovery sessions. The ALAD activity, as well as its gene expression, in the liver of the fatigued animals was decreased as compared with those of the control animals. Both activity and gene expression of ALAD were recovered to their respective control levels after the rats had been allowed to rest in their normal cages for 1 day. Furthermore, the activity of ALA synthase (ALAS), the rate-limiting enzyme in the heme biosynthesis, in the liver was increased after the fatigue session for 5 days. Although this level of increase in the plasma concentration of ALA may not induce fatigue, increase in plasma and urinary ALA levels can be biomarkers of fatigue.     

Prostate Cancer Associated Lipid Signatures in Serum Studied by ESI-Tandem Mass Spectrometryas Potential New Biomarkers

Prostate cancer (PCa) is one amongst the most common cancersin western men. Incidence rate ofPCa is on the rise worldwide. The present study deals with theserum lipidome profiling of patients diagnosed with PCa to identify potential new biomarkers. We employed ESI-MS/MS and GC-MS for identification of significantly altered lipids in cancer patient’s serum compared to controls. Lipidomic data revealed 24 lipids are significantly altered in cancer patinet’s serum (n = 18) compared to normal (n = 18) with no history of PCa. By using hierarchical clustering and principal component analysis (PCA) we could clearly separate cancer patients from control group. Correlation and partition analysis along with Formal Concept Analysis (FCA) have identified that PC (39:6) and FA (22:3) could classify samples with higher certainty. Both the lipids, PC (39:6) and FA (22:3) could influence the cataloging of patients with 100% sensitivity (all 18 control samples are classified correctly) and 77.7% specificity (of 18 tumor samples 4 samples are misclassified) with p-value of 1.612×10⁻⁶ in Fischer’s exact test. Further, we performed GC-MS to denote fatty acids altered in PCa patients and found that alpha-linolenic acid (ALA) levels are altered in PCa. We also performed an in vitro proliferation assay to determine the effect of ALA in survival of classical human PCa cell lines LNCaP and PC3. We hereby report that the altered lipids PC (39:6) and FA (22:3) offer a new set of biomarkers in addition to the existing diagnostic tests that could significantly improve sensitivity and specificity in PCa diagnosis.     

Metabonomic and microbiological analysis of the dynamic effect of vancomycin-induced gut microbiota modification in the mouse

The effects of the antibiotic vancomycin (2 x 100 mg/kg/day) on the gut microbiota of female mice (outbred NMRI strain) were studied, in order to assess the relative contribution of the gut microbiome to host metabolism. The host's metabolic phenotype was characterized using (1)H NMR spectroscopy of urine and fecal extract samples. Time-course changes in the gut microbiotal community after administration of vancomycin were monitored using 16S rRNA gene PCR and denaturing gradient gel electrophoresis (PCR-DGGE) analysis and showed a strong effect on several species, mostly within the Firmicutes. Vancomycin treatment was associated with fecal excretion of uracil, amino acids and short chain fatty acids (SCFAs), highlighting the contribution of the gut microbiota to the production and metabolism of these dietary compounds. Clear differences in gut microbial communities between control and antibiotic-treated mice were observed in the current study. Reduced urinary excretion of gut microbial co-metabolites phenylacetylglycine and hippurate was also observed. Regression of urinary hippurate and phenylacetylglycine concentrations against the fecal metabolite profile showed a strong association between these urinary metabolites and a wide range of fecal metabolites, including amino acids and SCFAs. Fecal choline was inversely correlated with urinary hippurate. Metabolic profiling, coupled with the metagenomic study of this antibiotic model, illustrates the close inter-relationship between the host and microbial "metabotypes", and will provide a basis for further experiments probing the understanding of the microbial-mammalian metabolic axis.     

Plasma catecholamines and urinary excretion of their main metabolites in three models of portal hypertension

We have measured, by a specific radioenzymoassay, the plasma concentration of dopamine (DA) and norepinephrine (NE) and by gas chromatography the urinary excretion of some catecholamine metabolites (HVA, homovanillic acid, DOPAC, dihydroxyphenyl acetic acid; VMA, vanilmandelic acid, and DOPEG, dihydroxyphenyl glycol) in three groups of rats with portal hypertension: cirrhotic rats (CR), rats with progressive portal hypertension (PPH) and rats with progressive hepatic congestion (PHC). The three groups of rats had portal hypertension. PPH and PHC had also intrahepatic hypertension. CR rats showed an increased urinary excretion of NE and DA metabolites with a normal plasma concentration of these catecholamines, suggesting an increased turnover of NE and DA in this experimental model. PPH animals had a high plasma DA concentration with a decreased urinary excretion of catecholamine metabolites. PHC showed high plasma DA and NE levels with normal or increased urinary excretion of its metabolites. These results suggest that an increased neural activity is present in the early stages of experimental cirrhosis in rats and this alteration does not seem directly related to the portal hypertension but perhaps to the intrahepatic hypertension or to the hepatocellular damage.     

Personal exposure to different levels of benzene and its relationships to the urinary metabolites S-phenylmercapturic acid and trans,trans-muconic acid

This report is part of an extensive study to verify the validity, specificity, and sensitivity of biomarkers of benzene at low exposures and assess their relationships with personal exposure and genetic damage. The study population was selected from benzene-exposed workers in Tianjin, China, based on historical exposure data. The recruitment of 130 exposed workers from glue-making or shoe-making plants and 51 unexposed subjects from nearby food factories was based on personal exposure measurements conducted for 3-4 weeks prior to collection of biological samples. In this report we investigated correlation of urinary benzene metabolites, S-phenylmercapturic acid (S-PMA) and trans,trans-muconic acid (t,t-MA) with personal exposure levels on the day of urine collection and studied the effect of dose on the biotransformation of benzene to these key metabolites. Urinary S-PMA and t,t-MA were determined simultaneously by liquid chromatography-tandem mass spectrometry analyses. Both S-PMA and t,t-MA, but specifically the former, correlated well with personal benzene exposure over a broad range of exposure (0.06-122 ppm). There was good correlation in the subgroup that had been exposed to <1 ppm benzene with both metabolites (P-trend <0.0001 for S-PMA and 0.006 for t,t-MA). Furthermore, the levels of S-PMA were significantly higher in the subgroup exposed to <0.25 ppm than that in unexposed subjects (n=17; P=0.001). There is inter-individual variation in the rate of conversion of benzene into urinary metabolites. The percentage of biotransformation of benzene to urinary S-PMA ranged from 0.005 to 0.3% and that to urinary t,t-MA ranged from 0.6 to approximately 20%. The percentage of benzene biotransformed into S-PMA and t,t-MA decreased with increasing concentration of benzene, especially conversion of benzene into t,t-MA. It appears that women excreted more metabolites than men for the same levels of benzene exposures. Our data suggest that S-PMA is superior to t,t-MA as a biomarker for low levels of benzene exposure.     

Immunosuppression in experimental cryptococcosis in rats: Modification of macrophage functions by T suppressor cells

The influence of lymphocytes on the modulation of macrophage functions in altered immune states induced by Cryptococcus neoformans infection in rats has been investigated. In this report we observed a decrease of in vitro phagocytic activity by peritoneal cells (PC) from rats that received T suppressor cells induced by cryptococcal infection, against both the same microorganism that stimulated this suppressor population (p<0.05) and another non-pathogenic primary yeast (Candida tropicalis), (p<0.02). The microbicide function of the PC from these animals present a significant decrease in challenge by C. tropicalis (p<0.002) when compared with PC from animals transferred with T normal cells. The transference of T suppressor cells induced by cryptococcal infection in animals immunized with human serum albumin-complete Freund's adjuvant (HSA-CFA) produces a significant alteration of the phagocytosis to HSA-human red cells (HSA-HRC) when compared with the phagocytosis observed in animals that received T normal cells or the phagocytosis of normal animals (p<0.001). We could also observe that the DTH to HSA studied during 30 days was negative in rats transferred with PC sensitizated with HSA and treated with suppressor T cells, when compared with the DTH response of animals transferred with PC-HSA cocultured with normal cells (p<0.05 21st day). The data presented in this paper illustrated that following infection of rats with C. neoformans there is a change in some population of accessory cells behavior reflected by the modification of several functions, such as phagocytosis, lytic activity and antigen presentation.     

Identification of urinary metabolites that distinguish membranous lupus nephritis from proliferative lupus nephritis and focal segmental glomerulosclerosis

Introduction

Systemic lupus erythematosus (SLE or lupus) is a chronic autoimmune disease, and kidney involvement with SLE, a.k.a. lupus nephritis (LN), is a frequent and severe complication of SLE that increases patient morbidity and mortality. About 50% of patients with SLE encounter renal abnormalities which, if left untreated, can lead to end-stage renal disease. Kidney biopsy is considered the criterion standard for diagnosis and staging of LN using the International Society of Nephrology/Renal Pathology Society (ISN/RPS) classification, which was developed to help predict renal outcomes and assist with medical decision-making. However, kidney biopsy-based classification of LN is highly invasive and impractical for real-time monitoring of LN status. Here, nuclear magnetic resonance (NMR) spectroscopy-based metabolic profiling was used to identify urinary metabolites that discriminated between proliferative and pure membranous LN as defined by the ISN/RPS classification, and between LN and primary focal segmental glomerulosclerosis (FSGS).

Methods

Metabolic profiling was conducted using urine samples of patients with proliferative LN without membranous features (Class III/IV; n = 7) or pure membranous LN (Class V; n = 7). Patients with primary FSGS and proteinuria (n = 10) served as disease controls. For each patient, demographic information and clinical data was obtained and a random urine sample collected to measure NMR spectra. Data and sample collection for patients with LN occurred around the time of kidney biopsy. Metabolic profiling analysis was done by visual inspection and principal component analysis.

Results

Urinary citrate levels were 8-fold lower in Class V LN compared to Class III/IV patients, who had normal levels of urinary citrate (P < 0.05). Class III/IV LN patients had > 10-fold lower levels of urinary taurine compared to Class V patients, who had mostly normal levels (P < 0.01). Class V LN patients had normal urinary hippurate levels compared to FSGS patients, who completely lacked urinary hippurate (P < 0.001).

Conclusions

This pilot study indicated differences in urinary metabolites between proliferative LN and pure membranous LN patients, and between LN and FSGS patients. If confirmed in larger studies, these urine metabolites may serve as biomarkers to help discriminate between different classes of LN, and between LN and FSGS.     

Effects of feeding and body weight loss on the 1H-NMR-based urine metabolic profiles of male Wistar Han rats: implications for biomarker discovery.

For almost two decades, 1H-NMR spectroscopy has been used as an 'open' system to study the temporal changes in the biochemical composition of biofluids, including urine, in response to adverse toxic events. Many of these in vivo studies have reported changes in individual metabolites and patterns of metabolites that correlated with toxicological changes. However, many of the proposed novel biomarkers are common to a number of different types of toxicity. These may therefore reflect non-specific effects of toxicity, such as weight loss, rather than a specific pathology. A study was carried out to investigate the non-specific effects on urinary metabolite profiles by administering four hepatotoxic compounds, as a single dose, to rats at two dose levels: hydrazine hydrate (0.06 or 0.08 g kg (1)), 1,2-dimethylhydrazine (0.1 or 0.3 g kg (-1)), alpha-napthylisothiocyanate (0.1 or 0.15 g kg(-1)) and carbon tetrachloride (1.58 or 3.16 g kg(-1)). The study included weight-matched control animals along with those that were dosed, which were then 'pair-fed' with the treated animals so they achieved a similar weight loss. The urinary metabolite profiles were investigated over time using 1H-NMR spectroscopy and compared with the pathology from the same animals. The temporal changes were analysed statistically using multivariate statistical data analysis including principal component analysis, partial least squares, parallel factor analysis and Fisher's criteria. A number of metabolites associated with energy metabolism or which are partially dietary in origin, such as creatine, creatinine, tricarboxylic acid (TCA) cycle intermediates, phenylacetylglycine, fumarate, glucose, taurine, fatty acids and N-methylnicotinamide, showed altered levels in the urine of treated and pair-fed animals. Many of these changes correlated well with weight loss. Interestingly, there was no increase in ketone bodies (acetate and beta-hydroxybutyrate), which might be expected if energy metabolism was switched from glycolysis to fatty acid beta-oxidation. In some instances, the metabolites that changed were considered to be non-specific markers of toxicity, but were also identified as markers of a specific type of toxicity. For example, taurine was raised significantly in carbon tetrachloride-treated animals but reduced in the pair-fed group. However, raised urinary bile acid levels were only seen after alpha-napthylisothiocyanate treatment. The methodology, statistical analysis used and the data generated will help improve the identification of specific markers or patterns of urinary markers of specific toxic effects.     

Metabolism of shikimic,quinic, and cyclohexanecarboxylic acids in germfree,conventional, and gnotobiotic rats

By using a new high-pressure liquid chromatography assay, the increase in urinary hipprate following ingestion of shikimic, quinic, and cyclohexanecarboxylic acid was studied to quantitate the extent of aromatization in germfree, gnotobiotic, and converitonal rats. Germfree rats aromatized 2% of a single dose of shikimic acid or quinic acid and 44% of cyclohexanecarboxylic acid. Conventional rats aromatized all three compounds; shikimic (12%), quinic (12%), and cyclohexanecarboxylic acid (61%). A human fecal flora was fed to otherwise germfree rats to determine the degree of association and the resulting effect upon the metabolism of shikimic, quinic, and cyclohexanecarboxylic acids in vivo. Following establishment of the human microflora and subsequent feedings of shikimic or quinic acids, excretion of urinary hippurate was five to seven times greater (10–15% of the dose) than in germfree rats fed the same acids. The results suggest that the intestinal flora is needed to metabolize the shikimic acid to substrate(s) (probably cyclohexanecarboxylic acid). This substrate can then be aromatized by mammalian enzymes.     

Excretion of multiple urinary biomarkers for radical induced damage in rats treated with three different nephrotoxic compounds

In the present study the urinary excretion of seven aldehydes, acetone and coproporphyrin III as non-invasive in vivo biomarkers of free radical damage was measured in rats after treatment with three nephrotoxic compounds: cisplatin, mercuric chloride (HgCl2) and N -acetyl- S -(1,1,2,2-tetrafluoroethyl)-L-cysteine (TFE-Nac). A clear difference between the different nephrotoxic compounds was found in the time interval between dosage and maximal toxicity, as measured by clinical chemical parameters in urine. In rats treated with TFE-Nac and HgCl2 this was fast: 12 h and 24 h after treatment, respectively. In the rats treated with cisplatin, however, nephrotoxicity occurred later: 96 h-108 h after treatment. Urinary creatinine excretion was decreased in all treatments. Therefore, the excretion of the proposed biomarkers was expressed as amount excreted per 12 h urine fraction as well as amount excreted per mol creatinine in each 12 h urine fraction. Urinary excretion of coproporphyrin III was decreased in almost all 12 h urine fractions with all treatments, however, when expressed per mol creatinine, increases were found in urine of rats treated with cisplatin and HgCl2. In cisplatin-treated rats an increase was found in the excretion of formaldehyde per 12 h, but acetaldehyde, propanal and MDA levels were decreased. Expressed per mol creatinine, MDA levels were decreased, but other aldehydes were increased. In HgCl2-treated rats urinary aldehyde excretion expressed per mol creatinine was increased. In TFE-Nac treated animals the urinary levels of acetaldehyde per 12 h were increased and per mol creatinine the levels of some aldehydes were only slightly increased. With none of the treatments did the increase in the biomarkers expressed per mol creatinine exceed the decrease in creatinine excretion. Similar time intervals were found between dosage and maximal excretion of biomarkers as for the time intervals between dosage and maximal toxicity. With all treatments significant increases in the excretion of acetone were found both per 12 h and per mol creatinine, probably related to the increased glucose excretion. It was concluded that no convincing evidence for free radical damage was found in the present study with the employed biomarkers.     

Excretion of multiple urinary biomarkers for radical induced damage in rats treated with three different nephrotoxic compounds

In the present study the urinary excretion of seven aldehydes, acetone and coproporphyrin III as non-invasive in vivo biomarkers of free radical damage was measured in rats after treatment with three nephrotoxic compounds: cisplatin, mercuric chloride (HgCl2) and N -acetyl- S -(1,1,2,2-tetrafluoroethyl)-L-cysteine (TFE-Nac). A clear difference between the different nephrotoxic compounds was found in the time interval between dosage and maximal toxicity, as measured by clinical chemical parameters in urine. In rats treated with TFE-Nac and HgCl2 this was fast: 12 h and 24 h after treatment, respectively. In the rats treated with cisplatin, however, nephrotoxicity occurred later: 96 h-108 h after treatment. Urinary creatinine excretion was decreased in all treatments. Therefore, the excretion of the proposed biomarkers was expressed as amount excreted per 12 h urine fraction as well as amount excreted per mol creatinine in each 12 h urine fraction. Urinary excretion of coproporphyrin III was decreased in almost all 12 h urine fractions with all treatments, however, when expressed per mol creatinine, increases were found in urine of rats treated with cisplatin and HgCl2. In cisplatin-treated rats an increase was found in the excretion of formaldehyde per 12 h, but acetaldehyde, propanal and MDA levels were decreased. Expressed per mol creatinine, MDA levels were decreased, but other aldehydes were increased. In HgCl2-treated rats urinary aldehyde excretion expressed per mol creatinine was increased. In TFE-Nac treated animals the urinary levels of acetaldehyde per 12 h were increased and per mol creatinine the levels of some aldehydes were only slightly increased. With none of the treatments did the increase in the biomarkers expressed per mol creatinine exceed the decrease in creatinine excretion. Similar time intervals were found between dosage and maximal excretion of biomarkers as for the time intervals between dosage and maximal toxicity. With all treatments significant increases in the excretion of acetone were found both per 12 h and per mol creatinine, probably related to the increased glucose excretion. It was concluded that no convincing evidence for free radical damage was found in the present study with the employed biomarkers.