Evaluation of Metabolic Changes in Acute Intermittent Porphyria Patients by Targeted Metabolomics.

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.

GC/MS in Recent Years Has Defined the Normal and Clinically Disordered Steroidome: Will It Soon Be Surpassed by LC/Tandem MS in This Role?

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.

Targeting human urinary metabolome by LC-MS/MS: a review.

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.

SULFATION PATHWAYS: Alternate steroid sulfation pathways targeted by LC-MS/MS analysis of disulfates: application to prenatal diagnosis of steroid synthesis disorders.

The steroid disulfates ( 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).

Evaluation of markers out of the steroid profile for the screening of testosterone misuse. Part II: Intramuscular administration.

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.

Evaluation of markers out of the steroid profile for the screening of testosterone misuse. Part I: Transdermal administration.

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.

Quantifying endogenous androgens, estrogens, pregnenolone and progesterone metabolites in human urine by gas chromatography tandem mass spectrometry.

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 β-glucuronidase activity only. After selecting adequate internal standards and choosing the optimal instrumental parameters, i.

2-picolylamine derivatization for high sensitivity detection of abscisic acid in apicomplexan blood-infecting parasites.

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.

Binge ethanol drinking during adolescence modifies cocaine responses in mice.

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.

Targeting tryptophan and tyrosine metabolism by liquid chromatography tandem mass spectrometry.

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.

Current LC-MS methods and procedures applied to the identification of new steroid metabolites.

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).

Chronic pain causes a persistent anxiety state leading to increased ethanol intake in CD1 mice.

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.

Derivatization of steroids in biological samples for GC-MS and LC-MS analyses.

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.

Ultra high performance liquid chromatography tandem mass spectrometric detection of glucuronides resistant to enzymatic hydrolysis: Implications to doping control analysis.

Controversial results have been reported in the literature regarding the behavior of two testosterone (T) metabolites (3α-glucuronide-6β-hydroxyandrosterone and 3α-glucuronide-6β-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.

Maternal separation induces neuroinflammation and long-lasting emotional alterations in mice.

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.

Mass spectrometric characterisation of a condensation product between porphobilinogen and indolyl-3-acryloylglycine in urine of patients with acute intermittent porphyria.

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).

Untargeted metabolomics in doping control: detection of new markers of testosterone misuse by ultrahigh performance liquid chromatography coupled to high-resolution mass spectrometry.

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.

Factors affecting urinary excretion of testosterone metabolites conjugated with cysteine.

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-17β-ol-3-one, and Δ(15) -AD; 15-androsten-3,17-dione) is evaluated for the first time.

Urinary cysteinyl progestogens: Occurrence and origin.

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.

Increased and mistimed sex hormone production in night shift workers.

Background: Night shift work has been associated with an increased risk for breast and prostate cancer. The effect of circadian disruption on sex steroid production is a possible underlying mechanism, underinvestigated in humans. We have assessed daily rhythms of sex hormones and melatonin in night and day shift workers of both sexes.

Detection and characterization of triamcinolone acetonide metabolites in human urine by liquid chromatography/tandem mass spectrometry after intramuscular administration.

Rationale: Glucocorticosteroids are prohibited in sports when used by systemic administrations (e.g. intramuscular, IM), whereas they are allowed using other ways of administration.

Formation of Δ(1) and Δ(6) testosterone metabolites by human hepatocytes.

The existence of urinary testosterone (T) metabolites conjugated with cysteine has been recently reported. The formation of a ring double bond by a phase I metabolic transformation and the subsequent nucleophilic conjugation with glutathione was proposed as a putative metabolic pathway for the occurrence of these metabolites in urine. The main goal of the present study was to confirm the first step of the postulated pathway.

Evaluation of the reporting level to detect triamcinolone acetonide misuse in sports.

Triamcinolone acetonide (TA) is prohibited in sport competitions using systemic administrations (e.g., intramuscular, IM), and it is allowed by other routes (e.

Circadian variation of melatonin, light exposure, and diurnal preference in day and night shift workers of both sexes.

Background: Light-at-night has been shown in experimental studies to disrupt melatonin production but this has only partly been confirmed in studies of night shift workers. In this cross-sectional study, we examined the circadian variation of melatonin in relation to shift status, individual levels of light-at-night exposure, and diurnal preference, an attribute reflecting personal preference for activity in the morning or evening.

Methods: One hundred and seventeen workers (75 night and 42 day) of both sexes, ages 22 to 64 years, were recruited from four companies.