High-resolution mass spectrometry applied to theidentification of transformation products of quinolones from stability studies and new metabolites of enrofloxacin in chicken muscle tissues

The aim of this work was the identification of new metabolites and transformation products (TPs) in chicken muscle from Enrofloxacin (ENR), Ciprofloxacin (CIP), Difloxacin (DIF) and Sarafloxacin (SAR), which are antibiotics that belong to the fluoroquinolones family. The stability of ENR, CIP, DIF and SAR standard solutions versus pH degradation process (from pH 1.5 to 8.0, simulating the pH since the drug is administered until its excretion) and freeze-thawing (F/T) cycles was tested. In addition, chicken muscle samples from medicated animals with ENR were analyzed in order to identify new metabolites and TPs. The identification of the different metabolites and TPs was accomplished by comparison of mass spectral data from samples and blanks, using liquid chromatography coupled to quadrupole time-of-flight (LC-QqToF) and Multiple Mass Defect Filter (MMDF) technique as a pre-filter to remove most of the background noise and endogenous components. Confirmation and structure elucidation was performed by liquid chromatography coupled to linear ion trap quadrupole Orbitrap (LC-LTQ-Orbitrap), due to its mass accuracy and MS/MS capacity for elemental composition determination. As a result, 21 TPs from ENR, 6 TPs from CIP, 14 TPs from DIF and 12 TPs from SAR were identified due to the pH shock and F/T cycles. On the other hand, 14 metabolites were identified from the medicated chicken muscle samples. Formation of CIP and SAR, from ENR and DIF, respectively, and the formation of desethylene-quinolone were the most remarkable identified compounds.

Tissue homing and persistence of defined antigen-specific CD8+ tumor-reactive T-cell clones in long-term melanoma survivors.

Tumor antigen-specific cytotoxic T cells (CTLs) play a major role in the adaptive immune response to cancers. This CTL response is often insufficient because of functional impairment, tumor escape mechanisms, or inhibitory tumor microenvironment. However, little is known about the fate of given tumor-specific CTL clones in cancer patients. Studies in patients with favorable outcomes may be very informative. In this longitudinal study, we tracked, quantified, and characterized functionally defined antigen-specific T-cell clones ex vivo, in peripheral blood and at tumor sites, in two long-term melanoma survivors. MAGE-A10-specific CD8+ T-cell clones with high avidity to antigenic peptide and tumor lytic capabilities persisted in peripheral blood over more than 10 years, with quantitative variations correlating with the clinical course. These clones were also found in emerging metastases, and, in one patient, circulating clonal T cells displayed a fully differentiated effector phenotype at the time of relapse. Longevity, tumor homing, differentiation phenotype, and quantitative adaptation to the disease phases suggest the contribution of the tracked tumor-reactive clones in the tumor control of these long-term metastatic survivor patients. Focusing research on patients with favorable outcomes may help to identify parameters that are crucial for an efficient antitumor response and to optimize cancer immunotherapy.

Determinants of the essential one-carbon metabolism metabolites, homocysteine, S-adenosylmethionine, S-adenosylhomocysteine and folate, in cerebrospinal fluid.

BACKGROUND: Disturbances in the levels of one-carbon (1C) metabolism metabolites have been associated with a wide variety of neuropsychiatric diseases. Cerebrospinal fluid (CSF) levels of homocysteine (Hcy) and the other 1C metabolites, nor their interrelatedness and putative determinants, have been studied extensively in a healthy population. METHODS: Plasma and CSF samples from 100 individuals free from neuropsychiatric diseases were analyzed (55 male, 45 female; age 50±17 years). In blood, we measured plasma Hcy, serum folate and serum vitamin B12. In CSF, we measured total Hcy, S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH) and 5-methyltetrahydrofolate (5-methylTHF). Highly selective analytical methods like liquid chromatography combined with either mass spectrometry or fluorescence detection were used. RESULTS: CSF Hcy was inversely correlated with CSF 5-methylTHF and positively with plasma Hcy, independent of serum folate status. CSF SAH correlated with age, lower CSF 5-methylTHF and higher CSF Hcy. CSF 5-methylTHF showed independent negative correlations with age and positive correlations with serum folate. CSF SAM did not correlate with any of the 1C metabolites. CONCLUSIONS: Aging is characterized by a reduction in CSF 5-methylTHF levels and increased CSF levels of the potentially neurotoxic transmethylation inhibitor SAH. CSF 5-methylTHF, which is itself determined in part by systemic folate status, is a powerful independent determinant of CSF levels of Hcy and SAH.

Reaktive Thymushyperplasie infolge der Therapie ACTH-produzierender Tumoren [Reactive thymus dysplasia following therapy for ACTH-producing tumors]

Surgical or conservative treatment of ACTH-producing tumors results in acute drop of the previously excessively high cortisol levels. The following associated pathophysiological changes also occur in the organism's recovery from stress, such as trauma, operation or chemotherapy of tumors. Both cases result in a regeneration of the immune system, which might even be exalted. The corresponding radiographic feature is the "rebound" enlargement of the thymus occurring about six months after remission of hypercortisolism. Histological examination reveals benign thymus hyperplasia. Especially in cases of still unknown primary tumor the appearance of this anterior mediastinal mass can lead to misdiagnosis. We present the cases of two patients with diffuse thymic hyperplasia following surgical and medical correction of hypercortisolism. One patient suffered from classic Cushing's disease responding to transsphenoidal resection of an ACTH-secreting pituitary microadenoma. Six months later CT of the chest incidentally demonstrated an anterior mediastinal mass known as thymic hyperplasia. The second patient presented with an ectopic, still unkown source of ACTH-production. Six months after medical correction of hypercortisolism CT of the thorax showed an enlargement of the anterior mediastinum. Thymectomy was performed in order to exclude thymus carcinoid. Histological examination revealed benign thymus hyperplasia with negative immunostaining. CONCLUSION: Radiologists and clinicians should be familiar with the pathophysiological changes resulting from precipitously dropping cortisol levels in order to prevent diagnostic errors and unnecessary operations.

In vitro negative selection of viral superantigen-reactive thymocytes by thymic dendritic cells.

Intrathymic expression of endogenous mouse mammary tumor virus (MMTV)-encoded superantigens (SAg) induces the clonal deletion of T cells bearing SAg-reactive T-cell receptor (TCR) Vbeta elements. However, the identity of the thymic antigen-presenting cells (APC) involved in the induction of SAg tolerance remains to be defined. We have analyzed the potential of dendritic cells (DC) to mediate the clonal deletion of Mtv-7-reactive TCR alphabeta P14 transgenic thymocytes in an in vitro assay. Our results show that both thymic and splenic DC induced the deletion of TCR transgenic double positive (DP) thymocytes. DC appear to be more efficient than splenic B cells as negatively selecting APC in this experimental system. Interestingly, thymic and splenic DC display a differential ability to induce CD4+ SP thymocyte proliferation. These observations suggest that thymic DC may have an important role in the induction of SAg tolerance in vivo.

Methods to Increase Durability of Reactive ("D" Cracking) Coarse Aggregate in Portland Cement Concrete, MLR-73-01, 1973

The coarse aggregates used for Portland Cement concrete in southwest Iowa have exhibited a poor serviceability. This early failure is attributed to a characteristic commonly referred as "D" cracking. "D" line cracking is a discolored area of concrete caused by many fine, parallel hairline cracks. "D" line cracking is primarily caused by the movement of water in and through coarse aggregate with a unique pore structure. The presence of the water in the aggregates at the time of freezing causes the "D" cracking to occur and early failure. By making the pore structure less permeable to moisture, it is thought the durability factor of the concrete should increase. By drying the aggregate before mixing and then mixing with the cement, the particles of cement should enter the outer pore structure, and upon hydration make the pore structure less permeable to moisture.

On-line desorption of dried blood spots coupled to hydrophilic interaction/reversed-phase LC/MS/MS system for the simultaneous analysis of drugs and their polar metabolites.

An assay for the simultaneous analysis of pharmaceutical compounds and their metabolites from micro-whole blood samples (i.e. 5 microL) was developed using an on-line dried blood spot (on-line DBS) device coupled with hydrophilic interaction/reversed-phase (HILIC/RP) LC/MS/MS. Filter paper is directly integrated to the LC device using a homemade inox desorption cell. Without any sample pretreatment, analytes are desorbed from the paper towards an automated system of valves linking a zwitterionic-HILIC column to an RP C18 column. In the same run, the polar fraction is separated by the zwitterionic-HILIC column while the non-polar fraction is eluted on the RP C18. Both fractions are detected by IT-MS operating in full scan mode for the survey scan and in product ion mode for the dependant scan using an ESI source. The procedure was evaluated by the simultaneous qualitative analysis of four probes and their relative phase I and II metabolites spiked in whole blood. In addition, the method was successfully applied to the in vivo monitoring of buprenorphine metabolism after the administration of an intraperitoneal injection of 30 mg/kg on adult female Wistar rat.

Reactive rise in blood pressure upon cuff inflation: cuff inflation at the arm causes a greater rise in pressure than at the wrist in hypertensive patients.

OBJECTIVE: Cuff inflation at the arm is known to cause an instantaneous rise in blood pressure, which might be due to the discomfort of the procedure and might interfere with the precision of the blood pressure measurement. In this study, we compared the reactive rise in blood pressure induced by cuff inflation when the cuff was placed at the upper arm level and at the wrist. PARTICIPANTS AND METHODS: The reactive rise in systolic and diastolic blood pressure to cuff inflation was measured in 34 normotensive participants and 34 hypertensive patients. Each participant was equipped with two cuffs, one around the right upper arm (OMRON HEM-CR19, 22-32 cm) and one around the right wrist (OMRON HEM-CS 19, 17-22 cm; Omron Health Care Europe BV, Hoofddorp, The Netherlands). The cuffs were inflated in a double random order (maximal cuff pressure and position of the cuff) with two maximal cuff pressures: 180 and 240 mmHg. The cuffs were linked to an oscillometric device (OMRON HEM 907; Omron Health Care). Simultaneously, blood pressure was measured continuously at the middle finger of the left hand using photoplethysmography. Three measurements were made at each level of blood pressure at the arm and at the wrist, and the sequence of measurements was randomized. RESULTS: In normotensive participants, no significant difference was observed in the reactive rise in blood pressure when the cuff was inflated either at the arm or at the wrist irrespective of the level of cuff inflation. Inflating a cuff at the arm, however, induced a significantly greater rise in blood pressure than inflating it at the wrist in hypertensive participants for both systolic and diastolic pressures (P<0.01), and at both levels of cuff inflation. The blood pressure response to cuff inflation was independent of baseline blood pressure. CONCLUSIONS: The results show that in hypertensive patients, cuff inflation at the wrist produces a smaller reactive rise in blood pressure. The difference between the arm and the wrist is independent of the patient's level of blood pressure.

Rapid LC-MS/MS quantification of the major benzodiazepines and their metabolites on dried blood spots using a simple and cost-effective sample pretreatment.

BACKGROUND: Dried blood spots (DBS) sampling has gained popularity in the bioanalytical community as an alternative to conventional plasma sampling, as it provides numerous benefits in terms of sample collection and logistics. The aim of this work was to show that these advantages can be coupled with a simple and cost-effective sample pretreatment, with subsequent rapid LC-MS/MS analysis for quantitation of 15 benzodiazepines, six metabolites and three Z-drugs. For this purpose, a simplified offline procedure was developed that consisted of letting a 5-µl DBS infuse directly into 100 µl of MeOH, in a conventional LC vial. RESULTS: The parameters related to the DBS pretreatment, such as extraction time or internal standard addition, were investigated and optimized, demonstrating that passive infusion in a regular LC vial was sufficient to quantitatively extract the analytes of interest. The method was validated according to international criteria in the therapeutic concentration ranges of the selected compounds. CONCLUSION: The presented strategy proved to be efficient for the rapid analysis of the selected drugs. Indeed, the offline sample preparation was reduced to a minimum, using a small amount of organic solvent and consumables, without affecting the accuracy of the method. Thus, this approach enables simple and rapid DBS analysis, even when using a non-DBS-dedicated autosampler, while lowering the costs and environmental impact.

Simultaneous determination of human plasma levels of citalopram, paroxetine, sertraline, and their metabolites by gas chromatography-mass spectrometry.

A gas chromatography-mass spectrometry method is presented which allows the simultaneous determination of the plasma concentrations of the selective serotonin reuptake inhibitors citalopram, paroxetine, sertraline, and their pharmacologically active N-demethylated metabolites (desmethylcitalopram, didesmethylcitalopram, and desmethylsertraline) after derivatization with the reagent N-methyl-bis(trifluoroacetamide). No interferences from endogenous compounds are observed following the extraction of plasma samples from six different human subjects. The standard curves are linear over a working range of 10-500 ng/mL for citalopram, 10-300 ng/mL for desmethylcitalopram, 5-60 ng/mL for didesmethylcitalopram, 20-400 ng/mL for sertraline and desmethylsertraline, and 10-200 ng/mL for paroxetine. Recoveries measured at three concentrations range from 81 to 118% for the tertiary amines (citalopram and the internal standard methylmaprotiline), 73 to 95% for the secondary amines (desmethylcitalopram, paroxetine and sertraline), and 39 to 66% for the primary amines (didesmethylcitalopram and desmethylsertraline). Intra- and interday coefficients of variation determined at three concentrations range from 3 to 11% for citalopram and its metabolites, 4 to 15% for paroxetine, and 5 to 13% for sertraline and desmethylsertraline. The limits of quantitation of the method are 2 ng/mL for citalopram and paroxetine, 1 ng/mL for sertraline, and 0.5 ng/mL for desmethylcitalopram, didesmethylcitalopram, and desmethylsertraline. No interferences are noted from 20 other psychotropic drugs. This sensitive and specific method can be used for single-dose pharmacokinetics. It is also useful for therapeutic drug monitoring of these three drugs and could possibly be adapted for the quantitation of the two other selective serotonin reuptake inhibitors on the market, namely fluoxetine and fluvoxamine.

Profiling of steroid metabolites after transdermal and oral administration of testosterone by ultra-high pressure liquid chromatography coupled to quadrupole time-of-flight mass spectrometry.

The screening of testosterone (T) misuse for doping control is based on the urinary steroid profile, including T, its precursors and metabolites. Modifications of individual levels and ratio between those metabolites are indicators of T misuse. In the context of screening analysis, the most discriminant criterion known to date is based on the T glucuronide (TG) to epitestosterone glucuronide (EG) ratio (TG/EG). Following the World Anti-Doping Agency (WADA) recommendations, there is suspicion of T misuse when the ratio reaches 4 or beyond. While this marker remains very sensitive and specific, it suffers from large inter-individual variability, with important influence of enzyme polymorphisms. Moreover, use of low dose or topical administration forms makes the screening of endogenous steroids difficult while the detection window no longer suits the doping habit. As reference limits are estimated on the basis of population studies, which encompass inter-individual and inter-ethnic variability, new strategies including individual threshold monitoring and alternative biomarkers were proposed to detect T misuse. The purpose of this study was to evaluate the potential of ultra-high pressure liquid chromatography (UHPLC) coupled with a new generation high resolution quadrupole time-of-flight mass spectrometer (QTOF-MS) to investigate the steroid metabolism after transdermal and oral T administration. An approach was developed to quantify 12 targeted urinary steroids as direct glucuro- and sulfo-conjugated metabolites, allowing the conservation of the phase II metabolism information, reflecting genetic and environmental influences. The UHPLC-QTOF-MS(E) platform was applied to clinical study samples from 19 healthy male volunteers, having different genotypes for the UGT2B17 enzyme responsible for the glucuroconjugation of T. Based on reference population ranges, none of the traditional markers of T misuse could detect doping after topical administration of T, while the detection window was short after oral TU ingestion. The detection ability of the 12 targeted steroids was thus evaluated by using individual thresholds following both transdermal and oral administration. Other relevant biomarkers and minor metabolites were studied for complementary information to the steroid profile, including sulfoconjugated analytes and hydroxy forms of glucuroconjugated metabolites. While sulfoconjugated steroids may provide helpful screening information for individuals with homozygotous UGT2B17 deletion, hydroxy-glucuroconjugated analytes could enhance the detection window of oral T undecanoate (TU) doping.

Proteasome-assisted identification of a SSX-2-derived epitope recognized by tumor-reactive CTL infiltrating metastatic melanoma.

The tumor Ag SSX-2 (HOM-MEL-40) was found by serological identification of Ags by recombinant expression cloning and was shown to be a cancer/testis Ag expressed in a wide variety of tumors. It may therefore represent a source of CD8(+) T cell epitopes useful for specific immunotherapy of cancer. To identify potential SSX-2-derived epitopes that can be recognized by CD8(+) T cells, we used an approach that combined: 1) the in vitro proteasomal digestion of precursor peptides overlapping the complete SSX-2 sequence; 2) the prediction of SSX-2-derived peptides with an appropriate HLA-A2 binding score; and 3) the analysis of a tumor-infiltrated lymph node cell population from an HLA-A2(+) melanoma patient with detectable anti-SSX-2 serum Abs. This strategy allowed us to identify peptide SSX-2(41-49) as an HLA-A2-restricted epitope. SSX2(41-49)-specific CD8(+) T cells were readily detectable in the tumor-infiltrated lymph node population by multimer staining, and CTL clones isolated by multimer-guided cell sorting were able to lyse HLA-A2(+) tumor cells expressing SSX-2.

Functional plasticity of the LACK-reactive Vbeta4-Valpha8 CD4(+) T cells normally producing the early IL-4 instructing Th2 cell development and susceptibility to Leishmania major in BALB / c mice.

Early production of IL-4 by LACK-reactive Vbeta4-Valpha8 CD4(+) T cells instructs aberrant Th2 cell development and susceptibility to Leishmania major in BALB / c mice. This was demonstrated using Vbeta4(+)-deficient BALB / c mice as a result of chronic infection with MMTV (SIM), a mouse mammary tumor virus expressing a Vbeta4-specific superantigen. The early IL-4 response was absent in these mice which develop a Th1 response to L. major. Here, we studied the functional plasticity of LACK-reactive Vbeta4-Valpha8 CD4(+) T cells using BALB/ c mice inoculated with L. major shortly after infection with MMTV (SIM), i. e. before deletion of Vbeta4(+) cells. These mice fail to produce the early IL-4 response to L. major and instead exhibit an IFN-gamma response that occurs within LACK-reactive Vbeta4-Valpha8 CD4(+) T cells. Neutralization of IFN-gamma restores the production of IL-4 by these cells. These data suggest that the functional properties of LACK-reactive Vbeta4-Valpha8 CD4(+) T cells are not irreversibly fixed.