Responsiveness to DDAVP in Cushing's disease is associated with USP8 mutations through enhancing AVPR1B promoter activity.

PURPOSE: To clarify the characteristics of Cushing's disease (CD) patients who respond to the desmopressin (DDAVP) test and its underlying mechanisms. METHODS: Forty-seven patients with CD who underwent DDAVP testing were included. Patients were divided into two groups: DDAVP test (+) (adrenocorticotropic hormone [ACTH] levels increased by ≥ 1.5-fold during the DDAVP test) and DDAVP test (-) (ACTH levels increased by < 1.5-fold). AVP receptor expression levels in these tumors were quantified using quantitative RT-PCR and immunohistochemistry. AVP receptor promoter activity was analyzed using a dual-luciferase reporter assay system. RESULTS: Females (96.9%) and USP8 mutants (85.7%) were more prevalent in the DDAVP test (+) than in the DDAVP test (-). Indeed, the ACTH and cortisol responsiveness to DDAVP was greater in USP8 mutation positive tumors than that in USP8 wild type tumors (3.0-fold vs. 1.3-fold, 1.6-fold vs. 1.1-fold, respectively). Responsiveness to DDAVP was correlated with the expression levels of AVPR1B, but not with those of AVPR2. Comparably, Avpr1b promoter activity was enhanced by the overexpression of mutant USP8 compared to the wild type. CONCLUSIONS: We found that the responsiveness of ACTH to DDAVP in CD was greater in tumors with USP8 mutations. The present data suggest that USP8 mutations upregulate the AVPR1B promoter activity. Additionally, we showed that the DDAVP test can predict the presence of USP8 mutations.

Recent improvements in sports drug testing concerning the initial testing for peptidic drugs (< 2 kDa) - sample preparation, mass spectrometric detection, and data review.

In this work, a novel initial testing assay based on liquid chromatography-mass spectrometry is presented, enabling the detection of peptidic drugs and drug candidates (< 2 kDa) prohibited in sports. The assay covers representatives and metabolites of gonadotropin releasing hormone and its analogs (GnRHs), growth hormone secretagogues (GHS), growth hormone releasing peptides (GHRPs), and the Vasopressin-analog Desmopressin. The general objective of this work was to reduce sample preparation efforts to a minimum while preserving highest possible sensitivity and specificity of the assay, demonstrating limits of detection between 50 and 200 pg/mL. Here, a "dilute-and-inject" strategy provides the simplest conceivable sample preparation procedure. Furthermore, the combination of well-established strategies for the determination of peptides, such as two-dimensional liquid chromatography, dimethyl sulfoxide (DMSO)-assisted electrospray ionization, high resolution mass spectrometric detection and a tailored reporter template, which facilitates data review enormously, provides a high-throughput initial testing assay for lower molecular mass peptidic and peptide-related analytes.

Inherited, congenital and acquired disorders by hemostasis (vascular, platelet & plasmatic phases) with repercussions in the therapeutic oral sphere

The hemostasis alterations, either congenital or hereditary origin, and acquired, are circumstances that hinder oral care to patients who suffer them and also generates in the professional who has to attend, high stress. Bleeding control once established and dental treatment planning, both in the aspect of preparation, as the realization of the odonto-stomatological therapeutic, has suffered updates that do need to remember certain aspects of the care of these patients. But we must not forget that the hematologist or internist who controls the patient's medical condition, is a cornerstone for the planning and implementation of treatment plans. We must also remember that, in certain circumstances, treatment should be performed in a hospital setting. In this review, we aim to provide the odonto-stomatologist guidance on how to address the problem and provide simple and updated guidelines to apply in the treatment of these people

No disponible

Large-molecule quantification: sensitivity and selectivity head-to-head comparison of triple quadrupole with Q-TOF.

BACKGROUND: Bioanalysts are continuously looking for innovative ideas or instruments to increase the sensitivity and selectivity of their assays. Research for better mass spectrometers is becoming crucial with the emerging trend of large-molecule quantification. This study lists the different advantages of high-resolution MS (HRMS) over standard triple quadrupole instruments and proposes basic guidelines on how to use HRMS for large-molecule quantification in a regulated environment. RESULTS: A direct comparison between HRMS and triple quadrupole instruments for the quantification of six different model peptides (desmopressin, calcitonin, enfuvirtide, exenatide, glucagon and somatostatin) was completed. The HRMS instrument, when used specifically for targeted quantification ('quant/quant'), showed equivalent or better sensitivity for all compounds tested. CONCLUSION: This paper demonstrates that the use of a HRMS instrument in a regulated environment is a viable technique for quantification of large molecules. The latter was able to allow flexibility and selectivity to adapt the specificity of each assay with sensitivity comparable to the triple quadrupole instrument.

[Is the DDAVP-test helpful to diagnose children?]. / Trägt ein DDAVP-Test bei Kindern zur Diagnose bei?

UNLABELLED: It is very difficult to determine if patients with a moderate low level of VWF parameters have mild disease or if they are just low normal (so called grey area of VWD). This applies particularly to pediatrics, because it is difficult to evaluate the bleeding history of children. Al our centres every child diagnosed with vWD gets DDAVP to test the response for it. This study was done to evaluate the DDAVP- test as a diagnostic tool. PATIENTS, METHODS: A retrospective analysis of data obtained with routine DDAVP administration for test purposes in 52 patients with borderline von Willebrand disease at the haemophilia centre Graz was done. The increase of VWF:Ag, VWF:RiCof and FVIII:C has been document and compared. RESULTS: All of our patients had a very good response after application of DDAVP. The increase of VWF:Ag, VWF:RiCof and FVIII:C was compared in patients with positive and negative bleeding anamneses. The patients with positive anamneses had significantly lower parameters at the beginning. The increase of VWF parameters did not differ significantly between the groups at the different time-points. These results demonstrate that a positive anamnesis is not significantly associated with a lower increase. On the other side a high increase is not associated with a negative anamnesis. CONCLUSION: It is not possible to use the DDAVP test as a diagnostic tool for patients within the diagnostic grey area of VWD.

LC-MS determination of desmopressin acetate in human skin samples.

A sensitive, selective and accurate high-performance liquid chromatography-mass spectroscopy (LC-MS) assay for the determination of desmopressin acetate (1-deamino-8-D-arginine vasopressin, DDAVP) from human skin samples was developed and validated. Pieces of human breast skin were impregnated with DDAVP solutions and DDAVP was extracted with an optimum extraction procedure. The extracted solutions were then analyzed by a LC system, comprising of a Nucleosil C18 column (CC 125/2, 120-3) and a mobile phase of 0.01% formic acid in a mixture of 1.6 mM ammonium acetate and acetonitrile (33:67, v/v), coupled with electrospray ionization mass spectrometry (ESI-MS). Satisfactory results were obtained with limits of detection and quantification as low as 10 and 40 ng/ml, respectively, and with very good intra- and inter-day repeatability.

N-trimethyl chitosan chloride as absorption enhancer in oral peptide drug delivery. Development and characterization of minitablet and granule formulations.

In this study, minitablet and granule formulations were developed as solid oral dosage forms for the delivery of peptide drugs with the absorption enhancer N-trimethyl chitosan chloride (TMC). Minitablets were deemed suitable as a dosage form due to their ability, as components of multiple unit dosage forms (MUDFs), to disperse from each other, before disintegration, effectively increasing the area in which the polymer can assert its absorption-enhancing effect. The polymer should be released from the dosage forms prior to the release of the peptide, which was, together with achieving maximum release of both ingredients, the main focus of this study. Desmopressin (1-(3-mercaptopropionic acid)-8-D-arginine vasopressin monoacetate (DDAVP) was used as model peptide drug. The optimized minitablet formulation consisted of two types of granules, namely DDAVP and TMC granules. DDAVP granules, containing tetraglycerol pentastearate (TGPS), were specifically aimed at delaying the release of the peptide from the dosage form. Burst release of TMC was attempted with TMC granules. Both these granule types were included in the granule formulation. Release profiles for both the optimized minitablet formulation as well as the granule formulation showed that the release of DDAVP was effectively delayed from the formulation compared to the formulation where no attempt at delaying the release was made. In comparison, more TMC was released, and at a faster rate, from the granule formulation than the optimized minitablet formulations. Both the optimized minitablet formulation and the granule formulation show suitable release profiles for the delivery of peptide drugs with TMC as absorption enhancer in solid oral dosage forms.

Determination of the content of desmopressin in pharmaceutical preparations by HPLC and validation of the method.

The aim of this study was to apply high performance liquid chromatography to the determination of content of desmopressin in pharmaceutical preparations and validation of the method. The satisfactory results have been obtained using a column Luna C 8.5 microm, 100 x 4.6 mm and a mobile phase containing 0.067 M phosphate buffer of pH = 7 and acetonitrile in the proportion 83:17. It has been shown that the elaborated method shows good precision and accuracy and can be applied to the qualitative and quantitative analysis of pharmaceutical preparations containing desmopressin.

Separation of selected peptides by capillary electroendoosmotic chromatography using 3 microns reversed-phase bonded silica and mixed-mode phases.

The retention behaviour and selectivity of selected basic, neutral and acidic peptides have been studied by capillary electroendoosmotic chromatography (CEC) with Hypersil C8, C18, Hypersil mixed-mode, and Spherisorb C18/SCX columns, 250 (335) mm x 100 microns, packed with 3 microns particles, and eluted with mobile phases composed of acetonitrile-triethylamine-phosphoric acid (TEAP) at pH 3.0 using a Hewlett-Packard Model HP3DCE capillary electrophoresis system. The selected peptides were desmopressin (D), two analogues (A and B) of desmopressin, oxytocin (O) and carbetocin (C). The peptides eluted either before or after the electroendoosmotic flow (EOF) marker, depending on the concentration of acetonitrile used and the buffer ionic strength. The retention and selectivity of these peptides under CEC conditions were compared to their behaviour in free zone capillary electrophoresis (CZE), where the separation mode was based on the electrophoretic migration of the analytes due to their charge and Stokes radius properties. In addition, their retention behaviour in RP-HPLC was also examined. As a result, it can be concluded that the elution process of this group of synthetic peptides in CEC with a TEAP buffer at pH 3.0 is mediated by a combination of both electrophoretic migration processes and retention mechanisms involving hydrophobic as well as silanophilic interactions. This CEC method when operated with these 3 microns reversed-phase and mixed-mode sorbents with peptides is thus a hybrid of two well-known analytical methods, namely CZE and RP-HPLC. However, the retention behaviour and selectivity of the selected peptides differs significantly in the CEC mode compared to the RP-HPLC or CZE modes. Therefore this CEC method with these peptides represents an orthogonal analytical separation procedure that is complimentary to both of these alternative techniques.

Peptide sequencing by partial acid hydrolysis and high resolution plasma desorption mass spectrometry.

A method of deriving peptide sequence information using partial acid hydrolysis in combination with accurate mass measurements and immonium ion analysis provided by high-resolution plasma desorption mass spectrometry has been developed. The technique is very simple in terms of the chemistry and involves a short-time (3-30 min) incubation of the peptide in 1N-6N HCl at 100-110 degrees C with subsequent mass spectrometric analysis. Partial acid hydrolysis is found to produce sequence-specific segments, often ladder-like, although not always a complete set. Two application examples of the method are given: the linear peptide bradykinin and desmopressin, a peptide with an internal S-S bond and a non-amino-acid constituent. The technique has proved to be particularly useful in cases where some a priori information on the peptide structure was already known or where the automated Edman degradation technique might yield erratic results or not work at all.

DDAVP treatment of diabetes insipidus during pregnancy and the post-partum period.

A 23 year old woman with diabetes insipidus who had previously been treated with pitressin, pituitary snuff and chlorpropamide, was treated with DDAVP during pregnancy. DDAVP concentrations immunoassayed as vasopressin were determined in maternal serum and breast milk. Oxytocin antibodies were also determined in maternal serum.

A standardized desmopressin test of renal concentrating ability.

Renal concentration tests were carried out on 45 healthy volunteers and 106 patients with chronic renal disease of moderate degree involving impaired concentrating ability. Each subject served as his own control. The control experiment involved a total of 36 hr of "dehydration" (no fluids per os or food with a high fluid content such as fruit) in which 4 hr clearance periods were started from the 12th and continued to thd 36th hr. The first 12 hr involved an overnight period from 20.00 hr. One week later the same subjects were given 10 micrograms demopressin (dDAVP) at the 13th hr and a subsequent 4 hr clearance period provided blood and urine samples to compare desmopressin-induced urine concentration with various stages of concentration during oral fluid withdrawal alone. The drug was given intranasally. We measured urine osmolality and concentrations of urea. Na, K and calculated the U/P creatinine concentration ratios and creatinine clearances (CCr). Using Uosm as the criterion, the dDAVP experiment at 12 hr gave the same results as 24 hr of fluid withdrawal alone. With the U/PCr ratio as the criterion, dDAVP + 12 hr gave the same results as 36 hr of fluid withdrawal alone. Between 32 and 36 hr dehydration, CCr decreased - otherwise it remained unchanged in both healthy and ill subjects, with and without dDAVP. The only side-effect was the discomfort of more than 12 hr dehydration. This would appear to simplify a potentially useful diagnostic and prognostic test.