Search

Objective: Most patients with Dravet syndrome (DS) have unremarkable neuroimaging studies. However, a small number of patients exhibit focal abnormalities that may modify the epilepsy phenotype. We report a case series of DS patients carrying SCN1A variants concurrent with additional focal brain lesions, aiming to provide details regarding their clinical course, electrographic findings, and imaging features., Methods: We reviewed the electronic medical records of patients with developmental and epileptic encephalopathies in our center, from January 2000 to December 2022, identifying 90 patients with DS resulting from SCN1A variants. Of these, patients displaying focal brain lesions were eligible., Results: Five patients (4 males and 1 female), with median age of 26 years, were included. All exhibited clinical and electroencephalographic features consistent with the DS spectrum. Sequencing analysis of the SCN1A gene identified pathogenic variants. Magnetic resonance imaging (MRI) revealed focal cortical dysplasia (FCD) in two patients, while the remaining three had cystic lesions. Three patients had previously undergone resective epilepsy surgery in other centers, with no improvement in seizure frequency. Neuropathology studies revealed the presence of FCD type IIA, intracranial teratomas, and dysembryoplastic neuroepithelial tumor (DNET)., Significance: When an individual with an established diagnosis of genetic epilepsy and a focal lesion on MRI is undergoing preoperative evaluation, it is crucial to conduct a comprehensive analysis to understand the relevance of the focal finding for the patient's phenotype and thus anticipate potential surgical outcomes. In instances where epilepsy in DS patients is influenced by a specific focal structural lesion, resective surgery should be carefully considered after precise pharmacological treatment, acknowledging the persistent influence of an SCN1A variant on expected outcomes., (© 2023 International League Against Epilepsy.)

Objectives: This retrospective case series study aimed to investigate the demographic and clinical patterns of primary stabbing headache (PSH). In addition, we tried to identify subgroups of treatment responses in a neurology outpatient consultation at a Portuguese tertiary hospital., Methods: Clinical records were retrospectively reviewed and patients meeting the International Classification of Headache Disorders, 3rd edition, criteria for PSH were identified from January 2014 to December 2020. We collected data regarding demographic characteristics, clinical features of the headache, primary headache comorbidities, and information about treatment-related do PSH., Results: Of 1857 patients, 32 (1.7%; mean [SD] age of onset 56 [3.5] years) had the final diagnosis of PSH. Regarding headache characteristics, 20 patients (62.5%) reported episodes of stabbing in fixed locations and 12 (37.5%) in multiple areas; the duration of each attack was between ≤5 s (seven [21.9%]), 5-60 s (20 [62.5%]), and ≥60 s (five [15.6%]). In all, 18 patients (56.3%) had an episodic course (vs. six of 32 [18.8%] an acute course and eight of 32 [25%] a chronic course). In all, 17 patients started medical treatment (53.1%), with total or partial improvement in 10 (58.8%) of them. It was found that patients with pain in fixed locations had a better response to treatment when compared to patients with multiple locations, in a statistically significant way (eight of 11 vs. two of six, p = 0.023)., Conclusion: In our sample, the mean age of onset of PSH was >50 years and there was a wide range of PSH duration. The duration of each attack (>5 s), the pain in fixed locations, non-daily episodes of the pain in each attack, and the intermittent course of headache were the most prevalent clinical features. Finally, patients with stabbing in localized areas had a better response to treatment., (© 2022 American Headache Society.)

Studies on steroid metabolism are of utmost importance to improve the detection capabilities of anabolic androgenic steroids (AASs) misuse in sports drug testing. In humans, glucuronoconjugates are the most abundant phase II metabolites of AAS. Bisglucuronidation is a reaction where two separated functional groups on the same molecule are conjugated with glucuronic acid. These metabolites have not been studied in depth for steroids and could be interesting markers for doping control. The aim of the present work was to study the ionization and collision-induced dissociation of steroid bisglucuronides to be able to develop mass spectrometric analytical strategies for their detection in urine samples after AAS administration. Because steroid bisglucuronides are not commercially available, 19 of them were qualitatively synthesized to study their mass spectrometric behavior. Bisglucuronides ionized as [M+NH 4 ] + in positive mode, and as [M-H] - and [M-2H] 2- in negative mode. The most specific product ions of steroid bisglucuronides in positive mode resulted from the neutral losses of 387 and 405 Da (corresponding to [M+NH 4 -NH 3 -2gluc-H 2 O] + and [M+NH 4 -NH 3 -2gluc-2H 2 O] + , respectively, being "gluc" a dehydrated glucuronide moiety), and in negative mode, the fragmentation of [M-2H] 2- showed ion losses of m/z 175 and 75 (gluc - and HOCH 2 CO 2 - , respectively). On the basis of the common behavior, a selected reaction monitoring method was developed to detect bisglucuronide metabolites in urine samples. As a proof of concept, urines obtained after administration of norandrostenediol were studied, and a bisglucuronide metabolite was detected in those urines. The results demonstrate the usefulness of the analytical strategy to detect bisglucuronide metabolites in urine samples, and the formation of these metabolites after administration of AAS., (Copyright © 2017 John Wiley & Sons, Ltd.)

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., (Copyright © 2015 John Wiley & Sons, Ltd.)

Glucocorticosteroids are prohibited in sports when used by systemic administrations (e.g. oral), whereas they are allowed using other administration ways. Strategies to discriminate between administrations routes have to be developed by doping control laboratories. For this reason, the metabolism of prednisolone (PRED) was studied using liquid chromatography coupled to tandem mass spectrometry. A single oral (10 mg) dose of PRED was administered to two healthy male volunteers. Urine samples were collected up to 6 days after administration. Samples were hydrolyzed with β-glucuronidase and subjected to liquid-liquid extraction with ethyl acetate in alkaline conditions. The extracts were analyzed by liquid chromatography coupled to tandem mass spectrometry. Precursor ion scan methods (m/z 77, 91, 105, 121, 147 and 171) in positive ionization and neutral loss scan methods (76 and 94 Da) in negative ionization modes were applied for the open detection of PRED metabolites. Using these methods, PRED parent compound plus 20 metabolites were detected. PRED and 11 metabolites were characterized by comparison with standards of the compounds (PRED, prednisone, 20β-dihydro-PRED and 20α-dihydro-PRED, 20β-dihydro-prednisone and 20α-dihydro-prednisone, 6β-hydroxy-PRED and 6α-hydroxy-PRED, 20β isomers and 20α isomers of 6β,11β,17α,20,21-pentahydroxypregnan-1,4-diene-3-one, 6α,11β,17α,20β,21-pentahydroxypregnan-1,4-diene-3-one and Δ(6) -PRED). Using mass spectrometric data, feasible structures were proposed for seven of the remaining nine detected metabolites, including several 6-hydroxy-metabolites. Eleven of the characterized metabolites have not been previously described. Maximum excretion rates for PRED metabolites were achieved in first 24 h; however, most of the metabolites were still detectable in the last collected samples (day 6)., (Copyright © 2015 John Wiley & Sons, Ltd.)

Different approaches for the non-target detection of corticosteroids in urine have been evaluated. As a result of previous studies about the ionization (positive/negative) and fragmentation of corticosteroids, several methods based on both precursor ion (PI) and neutral loss (NL) scans are proposed. The applicability of these methods was checked by the injection of a standard solution containing 19 model compounds. Five of the studied methods (NL of 76 Da; PI of 77, 91 and 105; PI of 237; PI of 121, 147 and 171; and NL of 38 Da) exhibited satisfactory results at the concentration level checked (corresponding to 20 ng/ml in sample). Some other methods in negative ionization mode such as the NL of 104 Da were found to lack sufficient sensitivity. Some of the applied methods were found to be specific for a concrete structure (NL of 38 Da for fluorine containing corticosteroids) while others showed a wide range applicability (PI of 77, 91 and 105 showed response in all model compounds). Interference by endogenous compounds was also tested by the analysis of negative urines and urines spiked with different corticosteroids. The suitability of these methods for the detection of corticosteroid metabolites was checked by the analysis of urine samples collected after the administration of methylprednisolone and triamcinolone. A combination of the reported methods seems to be the approach of choice in order to have a global overview about the excreted corticosteroid metabolites., (Copyright 2009 John Wiley & Sons, Ltd.)

An evaluation of derivatization procedures for the detection of beta(2)-agonists is presented. The study was performed with the beta(2)-agonists bambuterol, clenbuterol, fenoterol, formoterol, salbutamol, salmeterol and terbutaline. Different derivatizating agents were employed, aiming to obtain derivatives with high selectivity to be used in the gas chromatographic/mass spectrometric analysis of beta(2)-agonists in biological samples. Trimethylsilylation was compared with different agents and the role of some catalysts was evaluated. Acylation, combined trimethylsilylation and acylation, and the formation of cyclic methylboronates were also studied. Sterical hindrance caused by different substituents at the nitrogen atom of the beta-ethanolamine lateral chain of beta(2)-agonist molecules is mainly responsible for differences in the abundances of the derivatives obtained. The use of catalysts produces an increase in the derivatization yield, especially for compounds with low steric hindrance (substituents with primary and secondary carbon atoms). The formation of trimethylsilyl (TMS) ethers is not influenced by structural molecular differences when only hydroxy groups are involved in derivatization. Combined trimethylsilylation and acylation showed that compounds with a secondary carbon atom linked to the nitrogen atom form mainly N-TFA-O-TMS derivatives, with a small amount of N-TMS-O-TMS derivatives. Compounds with tert-butyl substituents at the amino group (bambuterol, salbutamol and terbutaline) formed O-TMS derivatives as the main products, although a limited amount of trifluoroacylation at the nitrogen atom also occurred. Cyclic methylboronates were formed with bambuterol, clenbuterol, formoterol, salbutamol and salmeterol. Owing to hydroxy substituents in unsuitable positions for ring formation, this procedure was not effective for fenoterol and terbutaline. Mass spectra of different derivatives and tentative fragmentation profiles are also shown. For screening purpose (e.g. sports drug testing), derivatization with MSTFA or BSTFA alone is recommended as a comprehensive derivatization technique for beta(2)-agonists owing to minimal by-product formation; formation of cyclic methylboronates can be useful for confirmation purposes. Detection limits were obtained for the TMS and cyclic methylboronate derivatives using the derivatizing reagents MSTFA and trimethylboroxine, respectively. For most of the compounds, lower detection limits were found for the TMS derivatives., (Copyright 2000 John Wiley & Sons, Ltd.)

We investigated the antinociceptive properties of dexketoprofen trometamol (S(+)-ketoprofen tromethamine salt; SKP), a new analgesic, antiinflammatory drug, using the pain-induced functional impairment model in the rat (PIFIR), an animal model of arthritic pain. SKP was compared with racemic ketoprofen tromethamine salt (rac-KP), R(-)-ketoprofen tromethamine salt (RKP), ketorolac (KET), and morphine (MOR). We also assessed the effects of flurbiprofen (rac-FB) and its enantiomers (SFB and RFB) in the same model. Groups of six rats received either vehicle or analgesic drug and antinociception was evaluated by evaluating the dose-response curves over time. SKP was an effective antinociceptive drug in this model and was almost equally potent by either oral or intracerebroventricular administration. The oral potency of SKP was similar to that of oral KET and greater than that of oral MOR. No significant differences were observed between racemic ketoprofen and its enantiomers when administered orally. In the rat, significant bioinversion of RKP to SKP occurs when RKP is given orally. After oral administration of RKP, SKP was detectable in 30 min and surpassed the concentration of RKP after 3 h. Nevertheless, when the compounds were given intracerebroventricularly, some stereoselectivity in favor of SKP was observed. Stereoselectivity was observed with flurbiprofen, an analogue of ketoprofen that does not undergo significant metabolic inversion. Whereas SFB was an effective antinociceptive, RFB had no antinociceptive effect at the doses tested when given either orally or intracerebroventricularly., (1998 American College of Clinical Pharmacology.)

We investigated the antinociceptive properties of dexketoprofen trometamol [S(+)-ketoprofen tromethamine salt; SKP], a new analgesic, antiinflammatory drug, using the pain-induced functional impairment model in the rat (PIFIR), an animal model of arthritic pain. SKP was compared with racemic ketoprofen tromethamine salt (rac-KP), R(-)-ketoprofen tromethamine salt (RKP), ketorolac (KET), and morphine (MOR). We also assessed the effects of flurbiprofen (rac-FB) and its enantiomers (SFB and RFB) in the same model. Groups of six rats received either vehicle or analgesic drug and antinociception was evaluated by evaluating the dose-response curves over time. SKP was an effective antinociceptive drug in this model and was almost equally potent by either oral or intracerebroventricular administration. The oral potency of SKP was similar to that of oral KET and greater than that of oral MOR. No significant differences were observed between racemic ketoprofen and its enantiomers when administered orally. In the rat, significant bioinversion of RKP to SKP occurs when RKP is given orally. After oral administration of RKP, SKP was detectable in 30 min and surpassed the concentration of RKP after 3 h. Nevertheless, when the compounds were given intracerebroventricularly, some stereoselectivity in favor of SKP was observed. Stereoselectivity was observed with flurbiprofen, an analogue of ketoprofen that does not undergo significant metabolic inversion. Whereas SFB was an effective antinociceptive, RFB had no antinociceptive effect at the doses tested when given either orally or intracerebroventricularly.

We used immunohistochemistry with monoclonal antibodies (TRPM1, TRPM2) and histochemistry (acid phosphatase (AcP)) to investigate the effects of cyclosporin A (CsA) on macrophages and interdigitating cells (IDCs) in adult rat thymus after 21 days of treatment, and 21 days after stopping treatment. We also studied the development of IDCs and macrophages in 2, 6, 12, 20 and 30-day-old rats after 21 days of CsA administration to the pregnant mothers. In adult rats after 21 days of CsA treatment, IDCs were absent and only a small number of macrophages were present in the cortex; 21 days after stopping treatment the distribution of IDCs and macrophages had become similar to that in normal adults. The AcP+ macrophages in treated adult rats disappeared, as shown by immunohistochemistry, 21 days after CsA treatment and were again present, similarly to control animals, 21 days after stopping treatment. Therefore CsA causes the thymus medulla of adult rats to disappear and also a significant decrease in the macrophage population. We also found that while in normal rat neonates the thymus has the features of the adult thymus by the 12th day, in neonates from CsA treated mothers this did not appear until the 30th day. CsA treatment to pregnant rats delays thymus development in the young animals but does not cause persisting morphological alterations. This last finding was similar to that observed in adult rats 21 days after the end of CsA treatment.