ABSTRACT
Acute intermittent porphyria (AIP) is an inherited rare hepatic disorder due to mutations within the hydroxymethylbilane gene. AIP patients with active disease overproduce aminolevulinic acid (ALA) and porphobilinogen (PBG) in the liver which are exported inducing severe neurological attacks. Different hepatic metabolic abnormalities have been described to be associated with this condition. The goal of this research was to explore the metabolome of symptomatic AIP patients by state-of-the art liquid chromatography-tandem mass spectrometry (LC-MS/MS). A case versus control study including 18 symptomatic AIP patients and 33 healthy controls was performed. Plasmatic levels of 51 metabolites and 16 ratios belonging to four metabolic pathways were determined. The results showed that the AIP patients presented significant changes in the two main areas of the metabolome under study: (a) the tryptophan/kynurenine pathway with an increase of tryptophan in plasma together with increase of the kynurenine/tryptophan ratio; and (b) changes in the tricarboxylic acid cycle (TCA) including increase of succinic acid and decrease of the fumaric acid/succinic acid ratio. We performed a complementary in vitro study adding ALA to hepatocytes media that showed some of the effects on the TCA cycle were parallel to those observed in vivo. Our study confirms in plasma previous results obtained in urine showing that AIP patients present a moderate increase of the kynurenine/tryptophan ratio possibly associated with inflammation. In addition, it also reports changes in the mitochondrial TCA cycle that, despite requiring further research, could be associated with an energy misbalance due to sustained overproduction of heme-precursors in the liver.
Subject(s)
Porphyria, Acute Intermittent , Aminolevulinic Acid/urine , Chromatography, Liquid , Humans , Kynurenine , Metabolomics , Succinic Acid , Tandem Mass Spectrometry , TryptophanABSTRACT
Gas chromatography/mass spectrometry (GC/MS) has been used for steroid analysis since the 1960s. The advent of protective derivatization, capillary columns, and inexpensive electron ionization bench-top single quadrupole soon made it the method of choice for studying disorders of steroid synthesis and metabolism. However, the lengthy sample workup prevented GC/MS from becoming routine for steroid hormone measurement, which was dominated by radioimmunoassay. It was the emergence of liquid chromatography/tandem MS (LC/MS/MS) that sparked a renewed interest in GC/MS for the multicomponent analysis of steroids. GC/MS is excellent at providing an integrated picture of a person's steroid metabolome, or steroidome, as we term it. We review the recent work on newly described disorders and discuss the technical advances such as GC coupling to triple quadrupole and ion trap analyzers, two-dimensional GC/MS, and alternative ionization and detection systems such as atmospheric pressure chemical ionization (APCI) and time of flight. We believe that no novel GC/MS-based technique has the power of GC(electron ionization)/MS/MS as a "discovery tool," although APCI might provide ultimate sensitivity, which might be required in tissue steroidomics. Finally, we discuss the role of LC/MS/MS in steroidomics. This remains a challenge but offers shorter analysis times and advantages in the detection and discovery of steroids with a known structure. We describe recent advances in LC/MS steroidomics of hydrolyzed and intact steroid conjugates and suggest the technique is catching up with GC/MS in this area. However, in the end, both techniques will likely remain complementary and both should be available in advanced analytical laboratories.
ABSTRACT
Urine is a biological matrix that contains hundreds of metabolic end products which constitute the urinary metabolome. The development and advances on LC-MS/MS have revolutionized the analytical study of biomolecules by enabling their accurate identification and quantification in an unprecedented manner. Nowadays, LC-MS/MS is helping to unveil the complexity of urine metabolome, and the results obtained have multiple biomedical applications. This review focuses on the targeted LC-MS/MS analysis of the urine metabolome. In the first part, we describe general considerations (from sample collection to quantitation) required for a proper targeted metabolic analysis. In the second part, we address the urinary analysis and recent applications of four relevant families: amino acids, catecholamines, lipids and steroids.
Subject(s)
Chromatography, Liquid/methods , Metabolomics/methods , Tandem Mass Spectrometry/methods , Urinalysis/methods , HumansABSTRACT
The steroid disulfates (aka bis-sulfates) are a significant but minor fraction of the urinary steroid metabolome that have not been widely studied because major components are not hydrolyzed by the commercial sulfatases commonly used in steroid metabolomics. In early studies, conjugate fractionation followed by hydrolysis using acidified solvent (solvolysis) was used for the indirect detection of this fraction by GC-MS. This paper describes the application of a specific LC-MS/MS method for the direct identification of disulfates in urine, and their use as markers for the prenatal diagnosis of disorders causing reduced estriol production: STSD (steroid sulfatase deficiency), SLOS (Smith-Lemli-Opitz syndrome) and PORD (P450 oxidoreductase deficiency). Disulfates were detected by monitoring a constant ion loss (CIL) from the molecular di-anion. While focused on disulfates, our methodology included an analysis of intact steroid glucuronides and monosulfates because steroidogenic disorder diagnosis usually requires an examination of the complete steroid profile. In the disorders studied, a few individual steroids (as disulfates) were found particularly informative: pregn-5-ene-3ß,20S-diol, pregn-5-ene-3ß,21-diol (STSD, neonatal PORD) and 5α-pregnane-3ß,20S-diol (pregnancy PORD). Authentic steroid disulfates were synthesized for use in this study as aid to characterization. Tentative identification of 5ξ-pregn-7-ene-3ξ,20S-diol and 5ξ-pregn-7-ene-3ξ,17,20S-triol disulfates was also obtained in samples from SLOS affected pregnancies. Seven ratios between the detected metabolites were applied to distinguish the three selected disorders from control samples. Our results show the potential of the direct detection of steroid conjugates in the diagnosis of pathologies related with steroid biosynthesis.
Subject(s)
Chromatography, Liquid/methods , Prenatal Diagnosis/methods , Steroids/biosynthesis , Sulfates/metabolism , Tandem Mass Spectrometry/methods , Gas Chromatography-Mass Spectrometry , HumansABSTRACT
Although the introduction by the World Anti-Doping Agency (WADA) of the steroid module of the athlete biological passport (ABP) marked an important step forward in the screening of testosterone (T) misuse, it still remains one of the most difficult challenges in doping control analysis. The urinary determination of alternative markers has been recently reported as a promising tool for improving the screening of T oral administration. However, their evaluation for other, commonly used, administration routes is still required. The main goal of this study is the evaluation of the potential of 2 groups of metabolites (cysteinyl conjugated and glucuronoconjugated) after transdermal and intramuscular administration of T. Their suitability was evaluated in individuals with both low basal (L-T/E) and medium basal (M-T/E) values of T/E. In this Part I, we evaluated the urinary excretion profile of these 2 groups of T metabolites after the administration of 3 doses of T gel to 12 volunteers (6 L-T/E and 6 M-T/E) for 3 consecutive days. For this purpose, 9 different concentration ratios (5 cysteinyl conjugated and 4 glucuronoconjugated markers) were studied. Both, the intra-individual variability and the detection windows (DW) obtained by each ratio were evaluated. Cysteinyl conjugates showed a general low intra-individual variability and DWs that were shorter than any other tested marker. Despite the relatively large intra-individual variability, the DWs reached by glucuronoconjugates (2-3 days) were similar to those obtained by markers currently included in the ABP. Overall; this evaluation advises for the introduction of additional glucuronoconjugated markers in the screening of transdermal T administration.
Subject(s)
Substance Abuse Detection/methods , Testosterone/urine , Administration, Cutaneous , Biomarkers/metabolism , Biomarkers/urine , Chromatography, High Pressure Liquid/methods , Doping in Sports , Gas Chromatography-Mass Spectrometry/methods , Humans , Limit of Detection , Male , Tandem Mass Spectrometry/methods , Testosterone/administration & dosage , Testosterone/metabolismABSTRACT
In the fight against doping, the introduction of alternative markers to the steroid profile can be considered as an effective approach to improve the screening capabilities for the detection of testosterone (T) misuse. The aim of this study was to evaluate the potential of several T metabolites (cysteinyl conjugated and glucuronoconjugated resistant to enzymatic hydrolysis) to detect both the transdermal and the intramuscular administration of T. In Part I of the study, we studied the potential of these metabolites for the detection of T transdermal administration. Results revealed that resistant glucuronides can be a suitable complement to the current steroid profile. In this, Part II, dedicated to the intramuscular administration, we studied the potential of cysteinyl conjugated, resistant glucuronoconjugated and 1-cyclopentenoylglycine (1-CPG) for the detection of a single intramuscular injection of T cypionate. Possible differences in the excretion profile of all markers were explored between individuals with low basal (n=6) and medium basal (n=6) values of the testosterone/epitestosterone ratio (T/E). The results showed that all tested markers presented low intra-individual stability in basal conditions. Despite this, all glucuronoconjugated markers and 1-CPG, but not the cysteinyl conjugated markers, provided detection windows that were similar or longer than those obtained by markers currently included in the steroid profile. Based on the results obtained from the 2 parts of this study and from previously reported data, the potential applicability and the limitations of including these markers in the steroid profile are discussed.
Subject(s)
Cysteine/urine , Glucuronides/urine , Glycine/analogs & derivatives , Substance Abuse Detection/methods , Testosterone/urine , Administration, Cutaneous , Biomarkers/urine , Cysteine/analogs & derivatives , Doping in Sports , Gas Chromatography-Mass Spectrometry/methods , Glycine/urine , Humans , Hydrolysis , Injections, Intramuscular , Male , Steroids/administration & dosage , Steroids/urine , Tandem Mass Spectrometry/methods , Testosterone/administration & dosageABSTRACT
BACKGROUND: Acute intermittent porphyria (AIP) is a rare metabolic disorder due to a deficiency of porphobilinogen deaminase, the third enzyme of the heme biosynthetic pathway. This low enzymatic activity may predispose to the appearance of acute neurological attacks. Seminal studies suggested that AIP was associated with changes in tryptophan homeostasis with inconclusive results. Therefore, the aim of this study was to analyze the urinary metabolome of AIP patients focusing on tryptophan metabolism using state-of-the-art technology. METHODS: This was a case-control study including a group of 25 AIP patients with active biochemical disease and increased excretion of heme-precursors and 25 healthy controls. Tryptophan and related compounds and metabolites including: large neutral amino acids (LNAAs), serotonin, kynurenine, kynurenic acid and anthranilic acid were quantified in urine by liquid chromatography tandem-mass spectrometry (LC-MS/MS). Twenty-nine biological markers (including metabolic ratios and absolute concentrations) were compared between patients and controls. RESULTS: Significant differences were found in the tryptophan-kynurenine metabolic pathway. Compared to controls, AIP patients showed: (a) increased urinary excretion of kynurenine and anthranilic acid (P<0.005); (b): elevation of the kynurenine/tryptophan ratio (P<0.001) and (c): decrease of the kynurenic acid/kynurenine ratio (P=0.001). In contrast, no differences were found in the serotonin metabolic pathway independently of the markers and ratios used. CONCLUSIONS: The results of the study demonstrate that there is an imbalance in the kynurenine metabolic pathway in AIP patients, with an increase of the kynurenine/tryptophan ratio in urine and a reduction of the kynurenic acid/kynurenine ratio. The modified ratios suggest induction of indoleamine 2,3-deoxygenase and decreased activity of kynurenine aminotransferase in the liver. The results confirm that LC-MS/MS is useful for the characterization of the urinary metabolome of hepatic porphyrias.
Subject(s)
Metabolome/physiology , Metabolomics/methods , Porphyria, Acute Intermittent/urine , Tryptophan/metabolism , Tryptophan/urine , Adult , Biomarkers/metabolism , Biomarkers/urine , Cohort Studies , Female , Humans , Kynurenine/metabolism , Male , Middle Aged , Porphyria, Acute Intermittent/metabolism , Serotonin/metabolism , Young AdultABSTRACT
We have developed a new liquid chromatography-electrospray ionization tandem mass spectrometry methodology based on 2-picolylamine derivatization and positive ion mode detection for abscisic acid (ABA) identification. The selected reaction leads to the formation of an amide derivative which contains a highly active pyridyl group. The enhanced ionization allows for a 700-fold increase over commonly monitored unmodified ABA, which in turn leads to excellent limits of detection and quantification values of 0.03 and 0.15ngmL-1, respectively. This method has been validated in the highly complex matrix of a red blood cell extract. In spite of the high sensitivity achieved, ABA could not be detected in Plasmodium falciparum-infected red blood cells, suggesting that, if present, it will be found either in ultratrace amounts or as brief bursts at defined time points within the intraerythrocytic cycle and/or in the form of a biosynthetic analogue.
Subject(s)
Abscisic Acid/analysis , Erythrocytes/metabolism , Malaria, Falciparum/metabolism , Picolinic Acids/chemistry , Plasmodium falciparum/metabolism , Erythrocytes/parasitology , Humans , Malaria, Falciparum/parasitology , Plant Growth Regulators/analysisABSTRACT
A method for the quantitation of 22 urinary steroids (androgens, estrogens and the main pregnenolone and progesterone metabolites) by means of gas chromatography tandem mass spectrometry using a triple quadrupole analyzer has been developed. Two different enzymatic hydrolysis protocols were investigated; one capable of releasing steroids present as both sulfates and glucuronides (total fraction), and another with ß-glucuronidase activity only. After selecting adequate internal standards and choosing the optimal instrumental parameters, i.e. chromatographic separation and ion transition conditions, the method was fully validated using both hydrolysis protocols. The method was shown to be linear (r >0.99) in the range of endogenous concentrations for all studied steroids with extraction recoveries higher than 80%. The use of labeled internal standards allowed for both a correct quantification and the evaluation of the rate of deconjugation for sulfates and glucuronides in every sample. In general, the sensitivity of the method was suitable for the detection of the endogenous levels, with limits of quantification ranging from 0.1 to 20ng/mL. Accuracies ranging from 80% to 120%, and relative standard deviations below 25% in intra- and inter- assay experiments were found for most of the analytes. The applicability of the validated method was tested by quantifying twenty-two metabolites in 24-h urine samples collected from healthy individuals. The ranges for the excretion of steroids in the total and glucuronide fractions obtained with the new method were compared with those available in the literature. By comparing the figures in both fractions, an estimation of the percentage that the sulfation represents for each steroid was also calculated. The presence of side enzymatic activities and the utility of the method for clinical studies as well as for doping control analysis is discussed.
Subject(s)
Androgens/urine , Estrogens/urine , Gas Chromatography-Mass Spectrometry/methods , Metabolome , Pregnenolone/urine , Progesterone/urine , Tandem Mass Spectrometry/methods , HumansABSTRACT
Binge ethanol drinking is an emerging pattern of excessive consumption among adolescents and young adults. Repeated ethanol intoxication has negative consequences during critical periods of brain development. Therefore, binge ethanol intake represents a vulnerability factor that promotes subsequent manifestations of neuropsychiatric disorders. In this study, we investigated the effects of oral binge ethanol intake during adolescence on the subsequent effects of cocaine in C57BL/6 mice. Firstly, we evaluated the oral ethanol intake of two binge ethanol procedures with different ethanol concentrations (20% v/v versus 30%, v/v). The highest ethanol intake was found in mice exposed to the lower ethanol concentration (20% v/v). In a second experiment, mice exposed to binge ethanol procedure were evaluated to study the effects of cocaine on locomotor activity, behavioural sensitization, and the reinforcing effects of cocaine in the self-administration paradigm. Mice exposed to ethanol binging showed discrete detrimental effects in responses to cocaine in the different experiments evaluated. Our findings revealed that the pattern of binge ethanol consumption in adolescent mice here evaluated produced a weak facilitation of cocaine responses. The present study highlights the importance of interventions to limit the deleterious effects of binge ethanol drinking during adolescence.
Subject(s)
Alcohol Drinking/adverse effects , Behavior, Animal/drug effects , Binge Drinking/physiopathology , Cocaine/pharmacology , Ethanol/adverse effects , Age Factors , Animals , Locomotion/drug effects , Male , Mice , Mice, Inbred C57BL , Self Administration/methodsABSTRACT
An imbalance in tryptophan (Trp) and tyrosine (Tyr) metabolites is associated with neurological and inflammatory disorders. The accurate and precise measurement of these compounds in biological specimens is a powerful tool to understand the biochemical state in several diseases. In this study, a rapid, accurate and sensitive method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the targeted analysis of the metabolism of Trp and Tyr has been developed and validated. The method allows for the adequate quantification of Trp, Tyr and, eight Trp metabolites, three Tyr metabolites, together with four competitive large neutral amino acids. Serotonin, 5-hydroxyindoleacetic acid, kynurenine, kynurenic acid, dopamine, and homovanilic acid were among the targeted compounds. Sample preparation, chromatographic separation and mass spectrometric detection were optimized in human urine, human plasma and mice prefrontal cortex extracts. The method was shown to be linear (r>0.98) in the range of endogenous concentrations for all studied metabolites. In general, the limits of detection were suitable for the detection of the endogenous levels. Intra- and inter-assay precisions below 25% and accuracies ranging from 80 to 120% were found for most of the analytes. The use of labeled internal standards corrected the moderate matrix effect observed for some compounds. The applicability of the method was confirmed by analyzing urine samples collected from 13 healthy volunteers and comparing the results with previously established normal ranges. In addition, urine samples from two patients and a heterozygous carrier of a family with disturbed monoamine metabolism due to a loss of function mutation in the MAOA gene (X-linked) were analyzed and compared with samples from controls. All data together show the potential of the developed approach for targeted metabolomic studies.
Subject(s)
Chromatography, Liquid/methods , Tandem Mass Spectrometry/methods , Tryptophan/blood , Tryptophan/urine , Tyrosine/blood , Tyrosine/urine , Adult , Aged , Aggression , Animals , Disruptive, Impulse Control, and Conduct Disorders/metabolism , Female , Genetic Diseases, X-Linked/metabolism , Healthy Volunteers , Humans , Intellectual Disability/metabolism , Kynurenic Acid/analysis , Kynurenine/analysis , Male , Mice , Middle Aged , Monoamine Oxidase/deficiency , Monoamine Oxidase/metabolism , Prefrontal Cortex/metabolism , Serotonin/analysis , Young AdultABSTRACT
The implementation of the athlete steroidal passport in doping control analysis aims to detect intra-individual changes in the steroid profile related to the abuse of anabolic steroids. In this context, the study of intrinsic variations associated with each marker is of utmost importance. In the present work, the influence of several factors in the excretion of the recently reported testosterone metabolites conjugated with cysteine (Δ(1) -AED; 1,4-androstadien-3,17-dione, Δ(6) -AED; 4,6-androstadien-3,17-dione, Δ(6) -T; 4,6-androstadien-17ß-ol-3-one, and Δ(15) -AD; 15-androsten-3,17-dione) is evaluated for the first time. Degradation experiments at 37 °C proved that, although the cysteinyl moiety is released, the variation for urinary Δ(1) -AED/Δ(6) -AED, Δ(1) -AED/Δ(6) -T ratios is less than 30%. Moreover, freeze/thaw cycle testing resulted in RSDs values below 15% for all the analytes. Regarding infradian variability, moderate variations (below 40%) were observed. Additionally, notable alterations in the excretion of these compounds have been observed in the earliest stages of pregnancy. UGT2B17 polymorphism, responsible for the low T/E ratio found in some population, does not influence the excretion of cysteinyl compounds whereas the intake of exogenous substances (alcohol or 5α-reductase inhibitors) dramatically affects their excretion. The urinary concentrations of Δ(1) -AED, Δ(6) -AED, and Δ(15) -AD decreased (<50 %) after the ethanol intake, whereas after the administration of dutasteride, an important increase was observed for the concentrations of Δ(6) -AED, Δ(6) -T and Δ(15) -AD. Overall, the presented data describes the stability of the urinary cysteinyl steroids under the influence of many factors, proving their potential as suitable parameters to be included in the steroidal module of the athlete's biological passport.
Subject(s)
Anabolic Agents/urine , Cysteine/urine , Substance Abuse Detection/methods , Testosterone/urine , Adult , Anabolic Agents/metabolism , Cysteine/metabolism , Doping in Sports , Female , Gas Chromatography-Mass Spectrometry/methods , Humans , Limit of Detection , Male , Pregnancy , Tandem Mass Spectrometry/methods , Testosterone/metabolism , Young AdultABSTRACT
Early life experiences play a key role in brain function and behaviour. Adverse events during childhood are therefore a risk factor for psychiatric disease during adulthood, such as mood disorders. Maternal separation is a validated mouse model for maternal neglect, producing negative early life experiences that result in subsequent emotional alteration. Mood disorders have been found to be associated with neurochemical changes and neurotransmitter deficits such as reduced availability of monoamines in discrete brain areas. Emotional alterations like depression result in reduced serotonin availability and enhanced kynurenine metabolism through the action of indoleamine 2, 3-dioxygenase in response to neuroinflammatory factors. This mechanism involves regulation of the neurotransmitter system by neuroinflammatory agents, linking mood regulation to neuroinmunological reactions. In this context, the aim of this study was to investigate the effects of maternal separation with early weaning on emotional behaviour in mice. We investigated neuroinflammatory responses and the state of the tryptophan-kynurenine metabolic pathway in discrete brain areas following maternal separation. We show that adverse events during early life increase risk of long-lasting emotional alterations during adolescence and adulthood. These emotional alterations are particularly severe in females. Behavioural impairments were associated with microglia activation and disturbed tryptophan-kynurenine metabolism in brain areas related to emotional control. This finding supports the preeminent role of neuroinflammation in emotional disorders.
Subject(s)
Brain/metabolism , Emotions/physiology , Kynurenine/metabolism , Maternal Deprivation , Neuroimmunomodulation/physiology , Animals , Avoidance Learning/physiology , Body Weight , Brain/growth & development , Disease Models, Animal , Exploratory Behavior/physiology , Female , Male , Maternal Behavior/psychology , Mice , Motor Activity/physiology , Pain Threshold/physiology , Pain Threshold/psychology , Random AllocationABSTRACT
The study of the metabolism of steroids has a long history; from the first characterizations of the major metabolites of steroidal hormones in the pre-chromatographic era, to the latest discoveries of new forms of excretions. The introduction of mass spectrometers coupled to gas chromatography at the end of the 1960's represented a major breakthrough for the elucidation of new metabolites. In the last two decades, this technique is being complemented by the use of liquid chromatography-mass spectrometry (LC-MS). In addition of becoming fundamental in clinical steroid determinations due to its excellent specificity, throughput and sensitivity, LC-MS has emerged as an exceptional tool for the discovery of new steroid metabolites. The aim of the present review is to provide an overview of the current LC-MS procedures used in the quest of novel metabolic products of steroidal hormones and exogenous steroids. Several aspects regarding LC separations are first outlined, followed by a description of the key processes that take place in the mass spectrometric analysis, i.e. the ionization of the steroids in the source and the fragmentation of the selected precursor ions in the collision cell. The different analyzers and approaches employed together with representative examples of each of them are described. Special emphasis is placed on triple quadrupole analyzers (LC-MS/MS), since they are the most commonly employed. Examples on the use of precursor ion scan, neutral loss scan and theoretical selected reaction monitoring strategies are also explained.
Subject(s)
Chromatography, Liquid/methods , Mass Spectrometry/methods , Steroids/analysis , Steroids/metabolism , Animals , Chromatography, Liquid/instrumentation , Humans , Ions/analysis , Ions/metabolism , Mass Spectrometry/instrumentation , Metabolomics/instrumentation , Metabolomics/methodsABSTRACT
Mood disorders and chronic pain are closely linked, but limited progress has been made in understanding the role of chronic and neuropathic pain in the aetiopathogenesis of depression. To explore the pathological mechanisms that mediate the association between pain and depressive-like behaviours, we studied the time-dependent effect of neuropathic pain on the development of anxiety-like and despair behaviours in CD1 mice. We analysed behavioural data, neuroinflammation reactions and changes in neurotransmitter (glutamate and serotonin) levels in the mouse prefrontal cortex. Sciatic-operated mice displayed long-lasting anxiety-like and despair behaviours, starting 5 and 20 days after partial sciatic nerve ligation, respectively. Glutamatergic neurotransmission and IL-1ß cytokine expression were enhanced in the prefrontal cortex of mice with neuropathic pain. We found no change in serotonin metabolism, cytokine IL-6 or brain-derived neurotrophic factor levels. While sciatic-operated mice exposed to intermittent ethanol intake (20% v/v) using the drinking in the dark procedure consumed higher amounts of ethanol than sham-operated mice, thermal allodynia and despair behaviour were not attenuated by ethanol consumption. Our findings reveal an association between glutamatergic neurotransmission and pain-induced mood disorders, and indicate that moderate ethanol consumption does not relieve nociceptive and depressive behaviours associated with chronic pain in mice.
Subject(s)
Alcohol Drinking/epidemiology , Anxiety/etiology , Chronic Pain/complications , Ethanol/administration & dosage , Animals , Anxiety/psychology , Behavior, Animal , Brain-Derived Neurotrophic Factor/metabolism , Chronic Pain/psychology , Depression/etiology , Depression/psychology , Disease Models, Animal , Glutamic Acid/metabolism , Hyperalgesia/complications , Hyperalgesia/psychology , Interleukin-6/metabolism , Male , Mice , Prefrontal Cortex/metabolism , Serotonin/metabolism , Time FactorsABSTRACT
The determination of steroids in biological samples is essential in different areas of knowledge. MS combined with either GC or LC is considered the best analytical technique for specific and sensitive determinations. However, due to the physicochemical properties of some steroids, and the low concentrations found in biological samples, the formation of a derivative prior to their analysis is required. In GC-MS determinations, derivatization is needed for generating volatile and thermally stable compounds. The improvement in terms of stability and chromatographic retention are the main reasons for selecting the derivatization agent. On the other hand, derivatization is not compulsory in LC-MS analyses and the derivatization is typically used for improving the ionization and therefore the overall sensitivity achieved.
Subject(s)
Gas Chromatography-Mass Spectrometry , Mass Spectrometry , Steroids/analysis , Adrenal Cortex Hormones/analysis , Adrenal Cortex Hormones/chemistry , Adrenal Cortex Hormones/metabolism , Aldehydes/chemistry , Chromatography, High Pressure Liquid , Humans , Hydroxides/chemistry , Ketones/chemistry , Phenols/chemistry , Steroids/chemistry , Steroids/metabolismABSTRACT
Controversial results have been reported in the literature regarding the behavior of two testosterone (T) metabolites (3α-glucuronide-6ß-hydroxyandrosterone and 3α-glucuronide-6ß-hydroxyetiocholanolone) excreted after T administration. Due to their potential as biomarkers of T misuse, a UHPLC-MS/MS method for the direct quantification of these glucuronides was developed and validated. In addition, the main phase II metabolites of T that compose the steroid profile used for doping control purposes (glucuronides of T, epitestosterone, androsterone and etiocholanolone) were included. The method was found to be linear and with suitable LODs and LOQs for all metabolites. The average accuracies were between 86% and 120%, the RSDs for the intra- and inter-day precision were below 15% and 25% respectively. The method showed low matrix effect. Samples obtained before and after the administration of T were analyzed by both the developed UHPLC-MS/MS method and the GC-MS/MS method currently used by anti-doping laboratories. Relevant disagreements between the results obtained for 3α-glucuronide-6ß-hydroxyandrosterone and 3α-glucuronide-6ß-hydroxyetiocholanolone quantitation were observed. These markers seemed to be more suitable for the screening of T misuse when detected by UHPLC-MS/MS. These discrepancies were further investigated in 50 urine samples from healthy volunteers. The two methods gave highly correlated results for all metabolites that are currently included in the athlete's steroid profile confirming the reliability of the UHPLC-MS/MS method. However, the quantification of 3α-glucuronide-6ß-hydroxyandrosterone and 3α-glucuronide-6ß-hydroxyetiocholanolone, was only possible by using the UHPLC-MS/MS method since three interfering compounds were observed when performing the GC-MS/MS analysis with the most intense ion transitions. These results confirm the potential of the resistant glucuronides as biomarkers of T misuse. Additionally, they suggest that previously reported reference ranges for these metabolites should be reevaluated.
Subject(s)
Glucuronidase/metabolism , Glucuronides/urine , Performance-Enhancing Substances/urine , Chromatography, High Pressure Liquid , Doping in Sports/prevention & control , Escherichia coli K12/enzymology , Glucuronides/metabolism , Healthy Volunteers , Humans , Hydrolysis , Performance-Enhancing Substances/metabolism , Tandem Mass SpectrometryABSTRACT
We document the presence of a previously unknown species in the urine of patients with acute intermittent porphyria (AIP). The compound was fully characterised by liquid chromatography tandem mass spectrometry. Interpretation of both full spectrum acquisition and product ion spectra acquired in positive and negative ionisation modes by quadrupole time of flight MS allowed for the identification of a condensation product arising from porphobilinogen (PBG, increased in the urine of AIP patients) and indolyl-3-acryloylglycine (IAG, derived from indolylacrylic acid and present in human urine). The structure was unequivocally confirmed through comparison between the selected reaction monitoring chromatograms obtained from the urinary species and the condensation product qualitatively synthesised in the laboratory. Owing to the large amounts of both PBG and IAG in urine of AIP patients, the possible ex vivo formation of PBG-IAG in urine samples was evaluated. The product was spontaneously formed at room temperature, at 4 °C and even during storage at -20 °C when spiking a control sample with PBG. A positive correlation was found between PBG and PBG-IAG in samples collected from AIP patients. However, no correlation was found between PBG-IAG and IAG. Purified PBG-IAG did not form the characteristic chromogen after application of p-dimethylaminobenzaldehyde in HCl, thus suggesting that the current techniques used to measure PBG in urine of AIP patients based on Ehlrich's reaction do not detect this newly characterised PBG-IAG fraction.
Subject(s)
Biomarkers/urine , Glycine/analogs & derivatives , Mass Spectrometry/methods , Porphobilinogen/urine , Porphyria, Acute Intermittent/urine , Adult , Female , Glycine/urine , Humans , Male , Middle Aged , Young AdultABSTRACT
The use of untargeted metabolomics for the discovery of markers is a promising and virtually unexplored tool in the doping control field. Hybrid quadrupole time-of-flight (QTOF) and hybrid quadrupole Orbitrap (Q Exactive) mass spectrometers, coupled to ultrahigh pressure liquid chromatography, are excellent tools for this purpose. In the present work, QTOF and Q Exactive have been used to look for markers for testosterone cypionate misuse by means of untargeted metabolomics. Two different groups of urine samples were analyzed, collected before and after the intramuscular administration of testosterone cypionate. In order to avoid analyte losses in the sample treatment, samples were just 2-fold diluted with water and directly injected into the chromatographic system. Samples were analyzed in both positive and negative ionization modes. Data from both systems were treated under untargeted metabolomic strategies using XCMS application and multivariate analysis. Results from the two mass spectrometers differed in the number of detected features, but both led to the same potential marker for the particular testosterone ester misuse. The in-depth study of the MS and MS/MS behavior of this marker allowed for the establishment of 1-cyclopentenoylglycine as a feasible structure. The putative structure was confirmed by comparison with synthesized material. This potential marker seems to come from the metabolism of the cypionic acid release after hydrolysis of the administered ester. Its suitability for doping control has been evaluated.
Subject(s)
Chromatography, High Pressure Liquid , Metabolomics , Tandem Mass Spectrometry , Testosterone/analysis , Urinalysis/methods , Humans , Male , Molecular Structure , Reference Standards , Testosterone/chemistry , Testosterone/metabolismABSTRACT
The presence of two cysteinyl progestogens, 16-cysteinyl-progesterone (16-Cys-Prog) and 16-cysteinyl-pregnenolone (16-Cys-Preg), in human urine is described for the first time. Their occurrence was unequivocally confirmed by comparison with synthesized material by using mass spectrometric detectors. Several experiments were performed in order to clarify their origin. The adrenal origin of both 16-Cys-Prog and 16-Cys-Preg can be inferred from the increase in their concentrations after ACTH stimulatory test, together with their circadian variation similar to the one observed for cortisol. Moreover, the notable increase in excretions of 16-Cys-Prog during the luteal phase of the menstrual cycle points towards an ovarian production for this progestogen. However, the analysis of samples during the course of two pregnancies revealed that, in spite of the large amounts of progesterone produced during gestation, the human placenta lacks the capacity to make 16-Cys-Prog. The adrenal and ovarian origin has been further indicated by the absence of both metabolites in samples collected from a subject with bilateral adrenalectomy and hypogonadotrophyic hypogonadism. Regarding liver action, in vitro studies with hepatocytes and progesterone indicate that, although the liver is able to metabolize progesterone to 6-dehydroprogesterone, it has not the enzymatic machinery for the generation of 16-dehydroprogesterone. Taken together, these results open the possibility for a noninvasive test for the simultaneous evaluation of progesterone biosynthesis in different organs.