Urinary Time- or Dose-Dependent Metabolic Biomarkers of Aristolochic Acid-Induced Nephrotoxicity in Rats.

The metabolic mechanisms underlying aristolochic acid (AA)-induced nephrotoxicity are inconclusive. A Gas Chromatography-Mass Spectrometer (GC-MS)-based metabolomic study was performed to analyze urinary metabolites in AA-treated rats at different dosages (10, 20, and 40 mg/kg) and time points (2, 4, and 6 days). Serum blood urea nitrogen (BUN), creatinine, and kidney injury were significantly changed only on the 6th day in 40 mg/kg AA group, whereas metabolic alternation appeared even on the 2nd day in 10 mg/kg AA group. A total of 84 differential metabolites were identified in 40 mg/kg AA groups time-dependently and 81 in 10, 20, and 40 mg/kg AA groups dose-dependently (6 days) compared with control group. Eight metabolites were selected as potential metabolic biomarkers including methylsuccinic acid, nicotinamide, 3-hydroxyphenylacetic acid, citric acid, creatinine, uric acid, glycolic acid, and gluconic acid. Four of them were dose-dependently altered including methylsuccinic acid, citric acid, creatinine, and 3-hydroxyphenylacetic acid, which were defined as "early metabolic biomarker." The alteration of nicotinamide, uric acid, and gluconic acid was time- and dose-dependent, whereas the change of glycolic acid was time- or dose-independent. The latter 4 metabolites were defined as "late metabolic biomarker" because of the obvious reduction on the 6th day in 40 mg/kg AA group. In summary, the urinary metabolic alterations were more sensitive than conventional biomarkers of renal injury. The identified metabolites suggested pathways of energy metabolism, gut microbiota, and purine metabolism were associated with AA-induced nephrotoxicity time- or dose-dependently. Further investigation was warranted to determine the roles of the 8 potential metabolic biomarkers in AA-induced nephrotoxicity.

Study of urinary 2-{[2-(acetylamino-2-carboxyethyl]sulfanyl}butanedioic acid, a mercapturic acid of rats treated with maleic acid.

Maleic anhydride was reported illegally adulterated into starch to prepare traditional foods for decades in Taiwan. Maleic acid (MA), hydrolyzed from maleic anhydride, could cause kidney damages to animals. The potential health effects due to long-term MA exposures through food consumption have been of great concerns. Assessment of the dietary MA exposures could be very difficult and complicated. One of the alternatives is to analyze an MA-specific biomarker to assess the daily total MA intake. Therefore, this paper aimed to study the mercapturic acid of MA, 2-{[2-(acetylamino)-2-carboxyethyl]sulfanyl}butanedioic acid (MAMA), with our newly-developed isotope-dilution online solid-phase extraction liquid chromatography tandem mass spectrometry (ID-SPE-LC-MS/MS) method. MAMA was first synthesized, purified, and characterized with NMR to reveal two diastereomers and used for developing the analytical method. The method was validated to reveal excellent sensitivity with a LOD at 16.3ng/mL and a LOQ at 20.6ng/mL and used to analyze MAMA in urine samples collected from Sprague-Dawley rats treated with a single dose of 0mg/kg, 6mg/kg, and 60mg/kg (n=5) of MA through gavage. Our results show dose-dependent increases in urinary MAMA contents, and 70% MAMA was excreted within 12h with no gender differences (p>0.05). A half life of urinary MAMA was estimated at 6.8h for rat. The formation of urinary MAMA validates it as a chemically-specific biomarker for current MA exposure. Future study of MA metabolism in vivo will elucidate mechanisms of MAMA formation, and analysis of this marker in epidemiology studies could help to shed light on the causal effects of MA on human.

Determination of 12 potential nephrotoxicity biomarkers in rat serum and urine by liquid chromatography with mass spectrometry and its application to renal failure induced by Semen Strychni.

In previous nephrotoxicity metabonomic studies, several potential biomarkers were found and evaluated. To investigate the relationship between the nephrotoxicity biomarkers and the therapeutic role of Radix Glycyrrhizae extract on Semen Strychni-induced renal failure, 12 typical biomarkers are selected and a simple LC-MS method has been developed and validated. Citric acid, guanidinosuccinic acid, taurine, guanidinoacetic acid, uric acid, creatinine, hippuric acid, xanthurenic acid, kynurenic acid, 3-indoxyl sulfate, indole-3-acetic acid, and phenaceturic acid were separated by a Phenomenex Luna C18 column and a methanol/water (5 mM ammonium acetate) gradient program with a runtime of 20 min. The prepared calibration curves showed good linearity with regression coefficients all above 0.9913. The absolute recoveries of analytes from serum and urine were all more than 70.4%. With the developed method, analytes were successfully determined in serum and urine samples within 52 days. Results showed that guanidinosuccinic acid, guanidinoacetic acid, 3-indoxyl sulfate, and indole-3-acetic acid (only in urine) were more sensitive than the conventional renal function markers in evaluating the therapeutic role of Radix Glycyrrhizae extract on Semen Strychni-induced renal failure. The method could be further used in predicting and monitoring renal failure cause by other reasons in the following researches.

[Experimental study of dicholine succinate pharmacokinetics].

We have conducted for the first time an experimental study of pharmacokinetics of dicholine succinate (DCS) for different ways of its administration in rats The quantitative evaluation of DCS and its metabolites was performed by the radioactive isotope technique. Various parameters of DCS pharmacokinetics were estimated, including the dose dependence of drug content in the blood plasma, total bioavailability, distribution kinetics, and the main ways of DCS excretion.

Biochemical, molecular, and clinical characteristics of children with short chain acyl-CoA dehydrogenase deficiency detected by newborn screening in California.

BACKGROUND: Short-chain acyl-CoA dehydrogenase deficiency (SCADD) is an autosomal recessive inborn error of mitochondrial fatty acid oxidation with highly variable biochemical, genetic, and clinical characteristics. SCADD has been associated with accumulation of butyryl-CoA byproducts, including butyrylcarnitine (C4), butyrylglycine, ethylmalonic acid (EMA), and methylsuccinic acid (MS) in body fluid and tissues. Differences in genotype frequencies have been shown between patients diagnosed clinically versus those diagnosed by newborn screening. Moreover, while patients diagnosed clinically have a variable clinical presentation including developmental delay, ketotic hypoglycemia, epilepsy and behavioral disorders, studies suggest patients diagnosed by newborn screening are largely asymptomatic. Scant information is published about the biochemical, genetic and clinical outcome of SCADD patients diagnosed by newborn screening. METHODS: We collected California newborn screening, follow-up biochemical levels, and ACADS mutation data from September, 2005 through April, 2010. We retrospectively reviewed available data on SCADD cases diagnosed by newborn screening for clinical outcomes. RESULTS: During the study period, 2,632,058 newborns were screened and 76 confirmed SCADD cases were identified. No correlations between initial C4 value and follow-up biochemical markers (C4, EMA or MS levels) were found in the 76 cases studied. We found significant correlation between urine EMA versus MS, and correlation between follow-up C4 versus urine EMA. Of 22 cases where ACADS gene sequencing was performed: 7 had two or more deleterious mutations; 8 were compound heterozygotes for a deleterious mutation and common variant; 7 were homozygous for the common variant c.625G>A; and 1 was heterozygous for c.625G>A. Significant increases in mean urine EMA and MS levels were noted in patients with two or more deleterious mutations versus mutation heterozygotes or common polymorphism homozygotes. Clinical outcome data was available in 31 patients with follow-up extending from 0.5 to 60 months. None developed epilepsy or behavioral disorders, and three patients had isolated speech delay. Hypoglycemia occurred in two patients, both in the neonatal period. The first patient had concomitant meconium aspiration; the other presented with central apnea, poor feeding, and hypotonia. The latter, a c.625G>A homozygote, has had persistent elevations in both short- and medium-chain acylcarnitines; diagnostic workup in this case is extensive and ongoing. CONCLUSIONS: This study examines the largest series to date of SCADD patients identified by newborn screening. Our results suggest that confirmatory tests may be useful to differentiate patients with common variants from those with deleterious mutations. This study also provides evidence to suggest that, even when associated with deleterious mutations, SCADD diagnosed by newborn screening presents largely as a benign condition.

Decreased oxidative phosphorylation and PGAM deficiency in horses suffering from atypical myopathy associated with acquired MADD.

Earlier research on ten horses suffering from the frequently fatal disorder atypical myopathy showed that MADD (multiple acyl-CoA dehydrogenase deficiency) is the biochemical derangement behind atypical myopathy. From five horses that died as a result of this disease and seven healthy control horses, urine and plasma were collected ante mortem and muscle biopsies were obtained immediately post-mortem (2 patients and 7 control horses), to analyse creatine, purine and carbohydrate metabolism as well as oxidative phosphorylation. In patients, the mean creatine concentration in urine was increased 17-fold and the concentration of uric acid approximately 4-fold, compared to controls. The highest degree of depletion of glycogen was observed in the patient with the most severe myopathy clinically. In this patient, glycolysis was more active than in the other patients and controls, which may explain this depletion. One patient demonstrated very low phosphoglycerate mutase (PGAM) activity, less than 10% of reference values. Most respiratory chain complex activity in patients was 20-30% lower than in control horses, complex II activity was 42% lower than normal, and one patient had severely decrease ATP-synthase activity, more than 60% lower than in control horses. General markers for myopathic damage are creatine kinase (CK) and lactic acid in plasma, and creatine and uric acid in urine. To obtain more information about the cause of the myopathy analysis of carbohydrate, lipid and protein metabolism as well as oxidative phosphorylation is advised. This study expands the diagnostic possibilities of equine myopathies.

Simultaneous determination of guanidinosuccinic acid and guanidinoacetic acid in urine using high performance liquid chromatography/tandem mass spectrometry.

We present a method for the simultaneous determination of guanidinosuccinic acid (GSA) and guanidinoacetic acid (GAA) from urine by protein precipitation and liquid chromatography/tandem mass spectrometry. The chromatographic separation was performed using a cation exchange column with an elution gradient of 0.1 mM and 20 mM ammonium acetate buffers. GSA was detected with the mass spectrometer in negative ion mode monitoring at m/z 174.1, and GAA, creatinine, arginine, and homoarginine were in positive ion mode monitoring at m/z 118.1, 114.1, 175.1, and 189.1, respectively. As an internal standard, L-arginine-(13)C(6) hydrochloride and creatinine-d(3) (methyl-d(3)) were used. The calibration ranges were 0.50-25.0 µg mL(-1), and good linearities were obtained for all compounds (r>0.999). The intra- and inter-assay accuracies (expressed as recoveries) and precisions at three concentration levels (1.00, 5.00 and 25.0 µg mL(-1)) were better than 83.8% and 7.41%, respectively. The analytical performance of the method was evaluated by determination of the compounds in urine from male C57BL/J Iar db/db diabetes mellitus (DM) mice. The values of GSA and GAA corrected by the ratios of the individual compounds to creatinine were significantly increased in DM mice compared with control mice. These results indicated that the newly developed method was useful for determining urinary guanidino compounds and metabolites of arginine.

LC-NMR identification of a novel taurine-related metabolite observed in (1)H NMR-based metabonomics of genetically hypertensive rats.

This paper describes the LC-NMR spectroscopic identification of a novel urinary endogenous metabolite responsible for the signals, which were found as major contributors to the separation between genetically hypertensive rats (SHRSP) and normotensive control rats (WKY) in previous NMR-based metabonomic studies. Urine samples from 26-week-old normotensive rats were analyzed by an LC-NMR system equipped with a reversed-phase column having high retention ability for polar compounds. (1)H NMR spectra were continuously obtained in the on-flow mode, and the retention times of the unassigned signals in question were determined. Various two-dimensional spectra were subsequently measured for the fraction containing the unassigned signals under the stop-flow mode, which enables for a long accumulation resulting in the enhancement of signal-to-noise ratios. The candidate compound obtained from these LC-NMR data was synthesized, and the NMR and mass spectra were compared with those of the LC-NMR fraction. The unknown metabolite was identified as succinyltaurine from these experiments together with standard addition experiments. This novel metabolite, which is characteristic of the normotensive rats, is very interesting because it is structurally related to hypotensive taurine, and not substantially detected in the genetically hypertensive rats, which excreted more taurine than the normotensive rats. The biological and pathophysiological significances of succinyltaurine remain to be investigated.

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

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

Accumulation of methylguanidine and changes in guanidino compound levels in plasma, urine, and kidneys of furosemide-treated rats.

Antidiuresis and renal diseases alter the levels of guanidino compounds (GCs) in various tissues. Therefore, we hypothesized that diuresis could also disturb GC metabolism, storage, and elimination. In this study, rats were made diuretic to analyze GC levels in plasma, urine, and kidneys. Furosemide was chosen because of its wide use in various human pathologies. Rats were injected intraperitoneally 5 or 10 mg furosemide spread over a 24-hour cycle. Urine was collected over a period of 24 hours before and during furosemide treatment. Plasma was obtained from arterial blood. Renal zones were dissected. The GCs were determined by liquid chromatography. Five milligrams of furosemide provoked a significant increase in plasma and urine levels of GCs compared with those of the controls. The renal distribution and content of GCs were weakly modified by furosemide except for methylguanidine (MG). The level of MG was enhanced by 10 to 16 times in all renal zones. The MG level was 60% higher in renal zones of rats treated with 10 rather than 5 mg furosemide. The fractional excretion of MG was decreased by furosemide. Our data suggest that MG accumulation in kidney and plasma was caused by furosemide, which might induce MG synthesis, and that MG washout from tissue cells into urine by furosemide through the kidney may cause an increase in MG in the kidney.

Ethylmalonic encephalopathy: further clinical and neuroradiological characterization.

Ethylmalonic encephalopathy (EE) is a rare metabolic disorder with an autosomal recessive mode of inheritance that is clinically characterized by neuromotor delay, hyperlactic acidemia, recurrent petechiae, orthostatic acrocyanosis, and chronic diarrhea. Increased urinary levels of ethylmalonic acid and methylsuccinic acid are the main biochemical features of the disorder. We report on two patients affected by EE who showed different clinical and neuroradiological patterns. Patient 1 presented with a chronic clinical course characterized by very slow neuromotor deterioration, ataxia, and dysarthria. In contrast, patient 2 had an acute neonatal onset with severe neuromotor retardation, severe generalized hypotonia, and intractable seizures. Neuroradiological follow-up of patient 1 detected a diffuse hyperintensity on the T2 images at the basal ganglia which remained stable during a period of four years. Patient 2, in contrast, showed a rapid process of cerebral, and in part, cerebellar atrophy. On the basis of our observations, we reviewed the data published in the literature and tried to delineate the natural history of EE, which appears to be characterized by a wide spectrum of severity in the clinical course. No reports on neuroradiological follow-up of EE patients are available in the literature with which to compare our data. Finally, both patients showed a muscle COX deficiency. The pathogenetic implications of such a biochemical finding will be also discussed.

Determination of the site of glucuronidation in an N-(3,5-dichlorophenyl)succinimide metabolite by electrospray ionization tandem mass spectrometry following derivatization to picolinyl esters.

Derivatization using 3-pyridylcarbinol coupled with liquid chromatography electrospray ionization tandem mass spectrometry (LC/MS/MS) was used to characterize a novel Phase II metabolite of the nephrotoxic agricultural fungicide, N-(3,5-dichlorophenyl)succinimide (NDPS). A glucuronide conjugate of N-(3,5-dichlorophenyl)-2-hydroxysuccinamic acid (2-NDHSA) was identified in the urine from a rat dosed with [14C]NDPS. However, 2-NDHSA contains an aliphatic hydroxyl group and a carboxylic acid group, both of which are potential sites for glucuronidation. Mass spectrometry alone was unable to distinguish between these possibilities. Since the position of glucuronidation may be important in the mechanism of NDPS-induced nephrotoxicity, chemical derivatization in conjunction with mass spectrometry was used to characterize the glucuronide. The 2-NDHSA glucuronide conjugate was isolated from rat urine, derivatized with 3-pyridylcarbinol, and the derivatized metabolite was then analyzed by LC/MS/MS. Two known NDPS metabolites, 2-NDHSA and N-(3,5-dichlorophenyl)succinamic acid (NDPSA), were also isolated from rat urine and derivatized similarly. 3-Pyridinylcarbinol reacted rapidly with the carboxylic acid groups and formation of the picolinyl esters increased the ionization potential under positive ion conditions. The urinary glucuronide of 2-NDHSA was identified as an alcohol-linked glucuronide by examination of the molecular ions and the collision-induced dissociation (CID) product ion spectra of the derivatized products. When used in combination with mass spectrometry, derivatization of carboxylic acids with 3-pyridylcarbinol provided useful mass fragmentations and is a rapid way to obtain structural information about the position of glucuronidation of NDPS metabolites.

Infusion of plasma expanders may lead to unexpected results in urinary protein assays.

Overt proteinuria was detected in the urine of a potential kidney donor, ultimately leading to the refusal of the kidneys for transplantation purposes. Histological examination of the kidneys did not reveal any abnormalities. Searching for substances that could have interfered with the urinary total protein assay, the role of infused, modified gelatin plasma expanders was investigated. We therefore measured the concentration of protein before and after the addition of various artificial plasma expanders to urine. Only when Biuret reagent or Pyrogallol Red dye were used did we find elevated concentrations of protein. Other methods, including the turbidimetric assays, did not detect additional amounts of protein in the spiked urine. We conclude that the infusion of modified gelatin solutions may cause apparent proteinuria. This effect is not observed with starch-based plasma expanders. Clinical chemists and clinicians should be aware of this phenomenon and possibly repeat the analysis with a different technique.

Comparative disposition of the nephrotoxicant N-(3, 5-dichlorophenyl)succinimide and the non-nephrotoxicant N-(3, 5-difluorophenyl)succinimide in Fischer 344 rats.

Disposition of the nephrotoxicant N-(3,5-dichlorophenyl)succinimide (NDPS) was compared with that of a nontoxic analog, N-(3, 5-difluorophenyl)succinimide (DFPS). Male Fischer 344 rats were administered 0.2 or 0.6 mmol/kg [14C]NDPS or [14C]DFPS (i.p. in corn oil). Plasma concentrations were determined from blood samples obtained through the carotid artery. Urine samples were analyzed for metabolite content by HPLC. Rats were sacrificed at 3 h (DFPS) or 6 h (NDPS) and tissue radiolabel content and covalent binding were determined. [14C]NDPS-derived plasma radioactivity levels were 6- to 21-fold higher and peaked later than those from [14C]DFPS. Six hours after dosing, NDPS was 40% eliminated in the urine compared with approximately 90% for DFPS. By 48 h, only 67% of the NDPS dose was eliminated in urine. In contrast, DFPS excretion was virtually complete within 24 h. NDPS underwent oxidative metabolism to a slightly greater extent than DFPS. Distribution of [14C]NDPS-derived radioactivity into the kidneys was 3- to 6-fold higher than that into the liver or heart, and was more extensive than with [14C]DFPS. NDPS also covalently bound to plasma, renal, and hepatic proteins to a greater extent than DFPS. In summary, NDPS achieves higher tissue and plasma concentrations, covalently binds to a greater extent, and is eliminated more slowly than DFPS. Differences in the lipid solubility of NDPS metabolites and DFPS metabolites may help explain these results. The overall greater tissue exposure of NDPS and its metabolites may contribute to differential toxicity of these analogs.

Ethylmalonic and methylsuccinic aciduria in ethylmalonic encephalopathy arise from abnormal isoleucine metabolism.

Ethylmalonic encephalopathy (EE), an organic aciduria of unknown etiology characterized by developmental delay, hypotonia, and vascular instability associated with lactic acidemia and urinary excretion of ethylmalonic acid (EMA) and methylsuccinic acid (MSA), has been described in 11 patients. To test the possibility that the underlying biochemical defect involves isoleucine catabolism, we determined the response to oral L-isoleucine (IIe) load (150 mg/kg) in a 5-year-old girl with EE and in three healthy, age- and sex-matched controls. Following IIe load in the patient, there was accumulation of 2-methylbutyrylglycine (2-MBG) and a delayed and lower peak urinary excretion of tiglylglycine (TGL), suggesting a partial defect in 2-methyl-branched chain acylcoenzyme A dehydrogenase (2M-BCAD). In vitro measurements 2M-BCAD activity in cultured skin fibroblasts from patients with EE have been reported to be normal. Our results show that isoleucine is a source for the elevated EMA and MSA in patients with EE, and suggest a functional, possibly secondary, deficiency of activity of 2M-BCAD in vivo.

Biochemical and histopathological changes in nephrectomized mice.

Renal failure is characterized by the retention of nitrogenous metabolites such as urea, creatinine (CTN) and other guanidino compounds (GCs), uric acid, and hippuric acid, which could be related to the clinical syndrome associated with renal insufficiency. A model of renal failure has been developed in male C57BL x Swiss-Webster mice using nephrectomy (NX) and/or arterial ligation. A sham group (group A) and two nephrectomized groups, group B (one kidney removed) and group C (one kidney removed and ligation of the contralateral anterior artery branch), were studied. Ten days postsurgery, morphological and functional indices of renal failure were investigated. Nephrectomized mice manifested features of renal failure like polyuria and wasting. CTN clearance (CTN[Cl]) decreased by +/-26% in group B and +/-33% in group C as compared with the control values. Marked increases in the plasma concentration of guanidinosuccinic acid ([GSA] fourfold) and guanidine ([G] twofold) were observed in the experimental animals. CTN and alpha-keto-delta-guanidinovaleric acid (alpha-keto-delta-GVA) reached levels of, respectively, 1.5-fold and twofold those of controls. Urinary GSA excretion increased and guanidinoacetic acid (GAA) excretion decreased about twofold in group C. GSA increases (2.6-fold) were also observed in the brain in group C, in addition to a significant increase of G (2.5-fold) and gamma-guanidinobutyric acid ([GBA] 1.5-fold). Finally, the extent of NX was found to be 45.2% in group B and 71.4% in group C. Light microscopy revealed an expansion and increase in cellularity of the mesangium of the glomeruli, particularly in group C. A significant correlation (r = .574, P < .0001) was found between CTN(Cl) and the degree of NX as calculated from the remaining functional area. These data suggest that the model can be used as a tool for further pathophysiological and/or behavioral investigations of renal failure.

Central nervous system malformations in ethylmalonic encephalopathy.

Central nervous system malformations have been reported in a number of inherited enzyme defects. Ethylmalonic encephalopathy, an organic aciduria of unknown pathogenesis, has not been reported previously in association with brain or spinal cord malformations. We report on 2 sibs with confirmed ethylmalonic encephalopathy and malformations of the central nervous system; one with tethered cord, the other with cerebellar tonsillar ectopia (Chiari I malformation).