Endogenous Urinary Metabolites Research Articles

Tackling new psychoactive substances through metabolomics: UHPLC-HRMS study on natural and synthetic opioids in male and female murine models

Novel psychoactive substances (NPS) represent a broad class of drugs new to the illicit market that often allow passing drug-screening tests. They are characterized by a variety of structures, rapid transience on the drug scene and mostly unknown metabolic profiles, thus creating an ever-changing scenario with evolving analytical targets. The present study aims at developing an indirect screening strategy for NPS monitoring, and specifically for new synthetic opioids (NSOs), based on assessing changes in endogenous urinary metabolite levels as a consequence of the systemic response following their intake. The experimental design involved in-vivo mice models: 16 animals of both sex received a single administration of morphine or fentanyl. Urine was collected before and after administration at different time points; the samples were then analysed with an untargeted metabolomics LC-HRMS workflow. According to our results, the intake of opioids resulted in an elevated energy demand, that was more pronounced on male animals, as evidenced by the increase in medium and long chain acylcarnitines levels. It was also shown that opioid administration disrupted the pathways related to catecholamines biosynthesis. The observed alterations were common to both morphine and fentanyl: this evidence indicate that they are not related to the chemical structure of the drug, but rather on the drug class. The proposed strategy may reinforce existing NPS screening approaches, by identifying indirect markers of drug assumption.

Quantitative determination of potential urine biomarkers of respiratory illnesses using new targeted metabolomic approach

The diagnosis of asthma and chronic obstructive pulmonary disease (COPD) can be challenging due to the overlap in their clinical presentations in some patients. There is a need for a more objective clinical test that can be routinely used in primary care settings. Through an untargeted 1H NMR urine metabolomic approach, we identified a set of endogenous metabolites as potential biomarkers for the differentiation of asthma and COPD. A subset of these potential biomarkers contains 7 highly polar metabolites of diverse physicochemical properties. To the best of our knowledge, there is no liquid chromatography-tandem mass spectrometry (LC-MS/MS) method that evaluated more than two of the target metabolites in a single analytical run. The target metabolites belong to the families of monosaccharides, organic acids, amino acids, quaternary ammonium compounds and nucleic acids, rendering hydrophilic interaction liquid chromatography (HILIC) an ideal technology for their quantification. Since a clinical decision is to be made from patients data, a fully validated analytical method is required for biomarker validation. Method validation for endogenous metabolites is a daunting task since current guidelines were designed for exogenous compounds. As such, innovative approaches were adopted to meet the validation requirements. Herein, we describe a sensitive HILIC-MS/MS method for the quantification of the 7 endogenous urinary metabolites. Detection was achieved in the multiple reaction monitoring (MRM) mode with polarity switching, using quadrupole-linear ion trap instrument (QTRAP 6500) as well as single ion monitoring in the negative-ion mode. The method was fully validated according to the regulatory guidelines. Linearity was established between 6 and 21000 ng/mL and quality control samples demonstrated acceptable intra- and inter-day accuracy (85.7%–112%), intra- and inter-day precision (CV% <11.5%) as well as stability under various storage and sample processing conditions. To illustrate the method's applicability, the validated method was applied to the analysis of a small set of urine samples collected from asthma and COPD patients. Preliminary modelling of separation was generated using partial least square discriminant analysis (R2 0.752 and Q2 0.57). The adequate separation between patient samples confirms the diagnostic potential of these target metabolites as a proof-of-concept for the differentiation between asthma and COPD. However, more patient urine samples are needed in order to increase the statistical power of the analytical model.

A new metabolomics-based strategy for identification of endogenous markers of urine adulteration attempts exemplified for potassium nitrite.

Urine adulteration to circumvent positive drug testing represents a problem for toxicological laboratories. While creatinine is a suitable marker for dilution, detection of chemicals is often performed by dipstick tests associated with high rates of false positives. Several methods would be necessary to check for all possible adulterants. Untargeted mass spectrometry (MS) methods used in metabolomics should theoretically allow detecting concentration changes of any endogenous urinary metabolite or presence of new biomarkers produced by chemical adulteration. As a proof of concept study, urine samples from 10 volunteers were treated with KNO2 and analyzed by high-resolution MS. For statistical data evaluation, XCMSplus and MetaboAnalyst were used. Compound identification was performed by database searches using an in-house database, Chemspider, METLIN, HMDB, and NIST. Principle component analysis revealed clear separation between treated and untreated urine samples. In detail, 307 features showed significant concentration changes with fold changes greater than 2 (79 decreased; 228 increased). Mainly amino acids (e.g., histidine, methylhistidine, di- and trimethyllysine) and purines (uric acid) were detected in lower amounts. 5-HO-isourate was found to be formed as a new compound from uric acid and, e.g., imidazole lactate concentrations increased due to the breakdown of histidine. This metabolomics-based strategy allowed for a broad identification range of markers of urinary adulteration. More studies will be needed to investigate routine applicability of identified potential markers exploring urinary conditions of their formation and stability. Selected markers might then be integrated into routine MS screening procedures allowing for detection of adulteration within routine MS analysis. Graphical Abstract ᅟ.

Quantification of candidate prostate cancer metabolite biomarkers in urine using dispersive derivatization liquid–liquid microextraction followed by gas and liquid chromatography–mass spectrometry

A simple, rapid and sensitive method based on dispersive derivatization liquid–liquid microextraction (DDLLME) combined with gas chromatography–mass spectrometry (GC–MS) and liquid chromatography–mass spectrometry (LC–MS) was developed and validated for the determination of prostate cancer metabolite biomarkers, including sarcosine, alanine, leucine and proline, in human urine samples. Dispersive derivatization using isobutyl chloroformate has been successfully employed to identify the amino acids of interest in ngmL−1 concentrations. Under the optimum experimental conditions, the detection limits of the amino acids were in the range of 0.05–0.1ngmL−1. The enrichment factor and relative recovery for the target amino acids were in the range of 140–155 and 93.8–106%, respectively. The proposed method showed good linearity (correlation coefficients >0.997), and good intra-day (below 7%) and inter-day precision (below 10%). This protocol provides a rapid, simple, selective and sensitive tool to quantify sarcosine and endogenous urinary metabolite for prostate cancer diagnosis and for a screening test.

Hippurate: The Natural History of a Mammalian–Microbial Cometabolite

Hippurate, the glycine conjugate of benzoic acid, is a normal constituent of the endogenous urinary metabolite profile and has long been associated with the microbial degradation of certain dietary components, hepatic function and toluene exposure, and is also commonly used as a measure of renal clearance. Here we discuss the potential relevance of hippurate excretion with regard to normal endogenous metabolism and trends in excretion relating to gender, age, and the intestinal microbiota. Additionally, the significance of hippurate excretion with respect to disease states including obesity, diabetes, gastrointestinal diseases, impaired renal function, psychological disorders and autism, as well as toxicity and parasitic infection, are considered.

Urinary Study on the Biochemical Effect of Acupuncture on Monosodium Urate Crystals-Induced Acute Gouty Arthritis in Rats using 600MHz 1H NMR

Acupuncture may potentially be an alternative therapy for acute gouty arthritis, a common inflammation model with multiple pathogenic mechanisms. In order to investigate the biological process of acute gouty arthritis and the effect of acupuncture, 1 H nuclear magnetic resonance (NMR)-based metabonomics has been applied to acquire urine metabolom data. Acute gouty arthritis model rats were induced by monosodium urate (MSU) crystals. The acupuncture on ST-36 (Zusanli) and SP-6 (Sanyinjiao) acupoints was applied for nine consecutive days. Principal Components Analysis (PCA) and Partial Least Squares (PLS) have been used to compare the time course urine metabolic perturbations. The prominent changes in endogenous urinary metabolites in acute gouty arthritis rats comprised increased levels of citrate and succinate, and decrease of allantoin, lactate, formate, trimethyl-N-oxide (TMAO) and taurine on the 3 rd day after MSU injection; and on day 9, Citrate, succinate, and allantoin began to recover to the original level, whereas formate, taurine, TMAO and lactate further decreased, which showed their incompletely recovery. Acupuncture treatment appears to reverse the levels of most metabolites affected by acute gouty arthritis, however, with significant increase levels of acetate, acetoacetate, allantoin, creatine/creatinine, phenylacetylglycine (PAG), taurine and TMAO on day 3; all these metabolites began to recover to the original level on day 9 except for creatine/creatinine, which slightly increased, demonstrating the therapeutic effects of acupuncture on acute gouty arthritis. Our work shows the potential of an NMR-based metabonomic approach in the study of biological effects of acupuncture on acute gouty arthritis

High resolution 1H NMR spectroscopic studies of the metabolism and excretion of ampicillin in rats and amoxycillin in rats and man.

High resolution proton nuclear magnetic resonance (1H NMR) spectroscopy has been used to investigate the metabolism and urinary excretion of the aminopenicillins, ampicillin and amoxycillin, in rats and of amoxycillin in man. 1H NMR resonances of the aminopenicillins, together with those for their 5R, 6R and 5S, 6R penicilloic acids and diketopiperazine metabolites were detected, assigned and quantified in urine samples with the aid of spin-echo NMR techniques. The dimer of amoxycillin was detected in rat urine for the first time together with novel drug-related resonances assigned to amoxycillin carbamate. Quantitative 1H NMR spectroscopic results were consistent with HPLC and microbiological data considering that only single measurements were recorded. Due to the short analysis time and simple sample preparation, NMR was particularly useful for studying the metabolism of the aminopenicillins for which sample degradation poses analytical problems. The non-invasive character of 1H NMR spectroscopic analysis of urine also provided unique information on a reversible reaction between amoxycillin and bicarbonate, an endogenous urinary metabolite.

Use of Proton NMR for Determining Changes in Metabolite Excretion Profiles Induced by Dietary Changes in the Rat

1H NMR spectroscopy was used to compare the endogenous urinary metabolite profiles of groups of Alp: APfSD (Wistar-derived) and Zucker (obese negative) rats fed different diets. With one of the diets (Special Dietary Services), hippuric acid was found to be absent in urine from both strains of rat whilst another component, 3-(3 hydroxyphenyl) propionic acid (3-HPPA) was identified. Switching either strain of rat from this diet to an alternative (Harlan Teklad) resulted in the appearance of hippuric acid in the urine concomitantly with the disappearance of 3-HPPA. Oral administration of benzoic acid (200 mg kg−1) to animals fed the diet which did not produce urinary hippuric acid resulted in a massive increase in hippuric acid excretion in the urine indicating that the rats retained the capacity to synthesize this material. We conclude that the lack of hippuric acid precursors in the urine along with the presence of 3-HPPA is due to diet and not strain of animal.

Toxicometabolomics approach to urinary biomarkers for mercuric chloride (HgCl 2)-induced nephrotoxicity using proton nuclear magnetic resonance ( 1H NMR) in rats

The primary objective of this study was to determine and characterize surrogate biomarkers that can predict nephrotoxicity induced by mercuric chloride (HgCl 2) using urinary proton nuclear magnetic resonance ( 1H NMR) spectral data. A procedure for 1H NMR urinalysis using pattern recognition was proposed to evaluate nephrotoxicity induced by HgCl 2 in Sprague–Dawley rats. HgCl 2 at 0.1 or 0.75 mg/kg was administered intraperitoneally (i.p.), and urine was collected every 24 h for 6 days. Animals ( n = 6 per group) were sacrificed 3 or 6 days post-dosing in order to perform clinical blood chemistry tests and histopathologic examinations. Urinary 1H NMR spectroscopy revealed apparent differential clustering between the control and HgCl 2 treatment groups as evidenced by principal component analysis (PCA) and partial least square (PLS)-discriminant analysis (DA). Time- and dose-dependent separation of HgCl 2-treated animals from controls was observed by PCA of 1H NMR spectral data. In HgCl 2-treated rats, the concentrations of endogenous urinary metabolites of glucose, acetate, alanine, lactate, succinate, and ethanol were significantly increased, whereas the concentrations of 2-oxoglutarate, allantoin, citrate, formate, taurine, and hippurate were significantly decreased. These endogenous metabolites were selected as putative biomarkers for HgCl 2-induced nephrotoxicity. A dose response was observed in concentrations of lactate, acetate, succinate, and ethanol, where severe disruption of the concentrations of 2-oxoglutarate, citrate, formate, glucose, and taurine was observed at the higher dose (0.75 mg/kg) of HgCl 2. Correlation of urinary 1H NMR PLS-DA data with renal histopathologic changes suggests that 1H NMR urinalysis can be used to predict or screen for HgCl 2-induced nephrotoxicity .

1H NMR-based metabonomic investigation of tributyl phosphate exposure in rats

Tributyl phosphate (TBP) is a toxic organophosphorous compound widely used in many industrial applications, including significant usage in nuclear processing. The industrial application of this chemical is responsible for occupational exposure and environmental pollution. In this study, 1H NMR-based metabonomics has been applied to investigate the metabolic response to TBP exposure. Male Sprague-Dawley rats were given a TBP-dose of 15 mg/kg body weight, followed by 24 h urine collection, as was previously demonstrated for finding most of the intermediates of TBP. High-resolution 1H NMR spectroscopy of urine samples in conjunction with statistical pattern recognition and compound identification allowed for the metabolic changes associated with TBP treatment to be identified. Discerning NMR spectral regions corresponding to three TBP metabolites, dibutyl phosphate (DBP), N-acetyl-(S-3-hydroxybutyl)- l-cysteine and N-acetyl-(S-3-oxobutyl)- l-cysteine, were identified in TBP-treated rats. In addition, the 1H NMR spectra revealed TBP-induced variations of endogenous urinary metabolites including benzoate, urea, and trigonelline along with metabolites involved in the Krebs cycle including citrate, cis-aconitate, trans-aconitate, 2-oxoglutarate, succinate, and fumarate. These findings indicate that TBP induces a disturbance to the Krebs cycle energy metabolism and provides a biomarker signature of TBP exposure. We show that three metabolites of TBP, dibutylphosphate, N-acetyl-(S-3-hydroxybutyl)- l-cysteine and N-acetyl-(S-3-oxobutyl)- l-cysteine, which are not present in the control groups, are the most important factors in separating the TBP and control groups ( p < 0.0023), while the endogenous compounds 2-oxoglutarate, benzoate, fumarate, trigonelline, and cis-aconetate were also important ( p < 0.01).

1H NMR and UPLC-MSE Statistical Heterospectroscopy: Characterization of Drug Metabolites (Xenometabolome) in Epidemiological Studies

Statistical HeterospectroscopY (SHY) is a statistical strategy for the coanalysis of multiple spectroscopic data sets acquired in parallel on the same samples. This method operates through the analysis of the intrinsic covariance between signal intensities in the same and related molecular fingerprints measured by multiple spectroscopic techniques across cohorts of samples. Here, the method is applied to 600-MHz (1)H NMR and UPLC-TOF-MS (E) data obtained from human urine samples ( n = 86) from a subset of an epidemiological population unselected for any relevant phenotype or disease factor. We show that direct cross-correlation of spectral parameters, viz. chemical shifts from NMR and m/ z data from MS, together with fragment analysis from MS (E) scans, leads not only to the detection of numerous endogenous urinary metabolites but also the identification of drug metabolites that are part of the latent use of drugs by the population. We show previously unreported positive mode ions of ibuprofen metabolites with their NMR correlates and suggest the detection of new metabolites of disopyramide in the population samples. This approach is of great potential value in the description of population xenometabolomes and in population pharmacology studies, and indeed for drug metabolism studies in general.

A 1H NMR-based metabonomic study of urine and plasma samples obtained from healthy human subjects

The aim of the study was to assess the feasibility of metabonomics in clinical studies. A 1H nuclear magnetic resonance (NMR)-based metabonomic analysis was performed on plasma and urine samples obtained from a group of 12 healthy male subjects on two separate study days 14 days apart. The subjects were fed a standard diet and plasma and urine samples were obtained on both days. The 1H NMR spectra obtained for urine and plasma samples were analysed using principal components analysis (PCA) in order to generate metabonomic data. In plasma there was relatively little variability between subjects and study days. In the case of endogenous urinary metabolite profiles there was considerable inter-subject variability, but less intra-subject variation. In all subjects diurnal variation was seen with urine samples. This suggests the possibility to collect consistent metabonomics data in clinical studies.

Classification of toxin-induced changes in 1H NMR spectra of urine using an artificial neural network

NMR spectra of urine from rats treated with a range of liver, kidney and testicular toxins at various doses were measured and classified using neural network methods. Toxin-induced changes in the levels of 18 low molecular weight endogenous urinary metabolites were assessed using a simple semi-quantitative scoring system. These scores were used as input to an artificial neural network, the use of which has been explored as a means of predicting the class of toxin. With this limited data set, based only the level of the maximal changes of these 18 metabolites, the network was able to predict the class and hence target organ of the toxins. Renal cortical toxicity was well predicted as was liver toxicity. The few examples of renal medullary toxins in the data set resulted in relatively poor training of the network although correct classification was still possible.

Endogenous urinary metabolite excretion in the growing pig

Abstract Endogenous urinary total nitrogen (N) and urea N excretions in the growing pig were measured using either the protein-free or regression method. Eighteen entire male pigs (28 kg liveweight) were randomly and equally allocated between six levels of feeding of a barley diet. Mean daily urinary total N and urea N excretions were determined. The regression of urinary metabolite output on dietary N intake extrapolated to zero N intake gave endogenous urinary total N and urea N excretions of 144.6 and 51.5 mg/kg0.75 per day, respectively. Corresponding values for six of the pigs subsequently given a protein-free diet for 11 days, with endogenous excretions calculated over the final six days, were 163.3 and 62.2 mg/kg0.75 per day. The urinary creatinine excretion of the barley-fed pigs was not related to dietary N intake. Mean daily urinary creatinine excretion was 53.1 ± 3.3 mg/kg0.75 whereas that for pigs given the protein-free diet was 61.2 ± 2.0 mg/kg0.75. Although endogenous urinary metabolite loss...