The Impact of Revascularisation on Quality of Life in Chronic Mesenteric Ischemia.

Background: Chronic mesenteric ischemia (CMI) is characterized by long-standing abdominal symptoms due to insufficient mesenteric circulation. Data on the effect of revascularisation on quality of life (QoL) for CMI are scarce. This study is the first to evaluate the impact of revascularisation on quality of life. Methods: Seventy-nine patients with CMI or acute-on-chronic mesenteric ischemia (AoCMI) underwent an intervention of one or more mesenteric arteries between January 2010 and July 2012. QoL before and after intervention was measured with the EuroQol-5D. Preintervention questionnaires were of standard care. Postintervention data were obtained by resending a questionnaire to the patients between February and May 2013. To investigate the clinical relevance of our findings, the minimal clinically important difference (MCID) was used. Since there is no established MCID for CMI, we used the literature reference MCID of inflammatory bowel syndrome (IBS) of 0.074. Results: Fifty-five (69.6%) of 79 patients returned their questionnaire and 23 (29.1%) were completely filled out. There was a significant increase of the median EQ-index score from 0.70 to 0.81 (p=0.02) and a significant reduction of symptoms in the domains usual activities (34.4%) and pain/discomfort (32.3%). There was a significant improvement of 17% in overall current health condition (VAS) (p=0.001). The MCID between baseline and postoperative EQ-5D index score was 0.162, indicating a clinically relevant improvement of quality of life after revascularisation. Conclusion: Quality of life of CMI patients is improved after mesenteric artery revascularisation.

The C terminal tail of the histamine H2 receptor contains positive and negative signals important for signal transduction and receptor down-regulation.

To examine the role of the C terminal tail in H2 receptor regulation, three cDNAs, encoding truncated histamine H2 receptor mutants (H2T295, H2T307, and H2T341), were constructed and stably transfected in Chinese hamster ovary (CHO) cells. The amino acids before position 307 appear to be necessary for proper receptor transport or folding, as no detectable H2 receptor binding of the H2T295 was observed after transfection. Truncation of the C terminal tail by 51 amino acids (H2T307) did not affect the binding properties of H2 antagonists and histamine or histamine-induced signaling. Yet, removal of 17 amino acids generated a mutant receptor (H2T341), which was able to form a ternary complex but was unable to fully activate the Gs protein on histamine exposure. Agonist-induced but not the cyclic AMP-dependent H2 receptor down-regulation was more profound for the H2T307 receptor, indicating that different structural elements of the H2 receptor protein are involved in the cyclic AMP-dependent and independent pathways of H2 receptor down-regulation. Taken together, in this study we identified regions in the C terminal tail of the H2 receptor that act as positive and/or negative signals in H2 receptor signaling and down-regulation.

Inverse agonism of histamine H2 antagonist accounts for upregulation of spontaneously active histamine H2 receptors.

Histamine H2 receptors transfected in Chinese hamster ovary (CHO) cells are time- and dose-dependently upregulated upon exposure to the H2 antagonists cimetidine and ranitidine. This effect appears to be H2 receptor-mediated as no change in receptor density was observed after H1 or H3 antagonist treatment or after incubation with the structural analogue of cimetidine, VUF 8299, which has no H2 antagonistic effects. By using transfected CHO cells expressing different densities of wild-type H2 receptors or an uncoupled H2Leu124Ala receptor, the histamine H2 receptor was found to display considerable agonist-independent H2 receptor activity. Cimetidine and ranitidine, which both induce H2 receptor upregulation, actually functioned as inverse agonists in those cell lines displaying spontaneous agonist-independent H2 receptor activity. Burimamide, on the other hand, was shown to act as a neutral antagonist and did as expected not induce H2 receptor upregulation after long-term exposure. The displayed inverse agonism of H2 antagonists appears to be a mechanistic basis for the observed H2 antagonist-induced H2 receptor upregulation in transfected CHO cells. These observations shed new light on the pharmacological classification of the H2 antagonists and may offer a plausible explanation for the observed development of tolerance after prolonged clinical use.

Identification of ligand binding residues in extracellular loops of the melanocortin 1 receptor.

To investigate whether residues in the extracellular domains of melanocortin 1 receptor (MC1R) are required for ligand binding, a number of mutants were constructed where charged residues were converted to alanine. The residues targeted for mutagenesis were Ser6, Glu102, Arg109, Asp184, Glu269, and Thr272. The mutant receptor DNAs were transiently expressed in COS-1 cells and their ability to bind [N1e4,D-Phe7]-alpha-MSH (NDP-MSH) was evaluated. Substitution of Asp184 by alanine completely abolished the binding of radiolabelled NDP-MSH as well as ACTH, even though the mutated receptor could be detected on cell surface using anti MC1R specific polyclonal antiserum. Mutations of Ser6, Glu269 and Thr272 resulted in a considerable loss of affinity for radiolabelled NDP-MSH as well as the ability of alpha-MSH to displace the bound radiolabelled NDP-MSH. The results demonstrate that the extracellular loops of human MC1R contain important ligand binding epitopes.

Visualization of agonist-induced internalization of histamine H2 receptors.

Histamine H2 receptors were tagged at the N-terminus with the eight amino acid Flag epitope to allow the immunological identification of the receptor peptide with the monoclonal anti-Flag M2 antibody. The introduction of the epitope did not modify the binding of several H2 ligands to the H2 receptor, nor the ability of histamine to stimulate the H2 receptor mediated cAMP production in HEK-293 cells. Western blots revealed a major protein band of 57 +/- 1 kDa, whereas a second band of 31 +/- 1 kDa was probably the result of a proteolytic breakdown of the 57 kDa band. Immunofluorescence measurements of stably transfected HEK-293 cells revealed the presence of anti-Flag-immunoreactivity in the plasma membrane. This immunoreactivity completely disappeared after a one hour treatment with histamine. The receptor internalization was reversible and blocked by the endocytosis inhibitor phenylarsine oxide. Forskolin did not induce H2 receptor internalization, indicating that histamine causes H2 receptor internalization via a cAMP-independent pathway.

Histamine as a marker for hydroxyl radicals.

During inflammation an influx of neutrophils and release of mediators from mast cells (such as histamine) take place. The stimulated neutrophils can produce reactive oxygen species (ROS). One of these ROS is the highly reactive hydroxyl radical (OH(.)). It would be interesting to be able to quantify the extent of ROS formation. We investigated if histamine which is present at the inflammation site can serve as an endogenous marker for the formation of OH(.). We found that histamine after incubation with OH(.) gave two distinct products in our HPLC system. One of the products gave the same characteristics as the synthesized 2-imidazolone derivative of histamine. This suggests that this derivative will be formed when histamine is incubated with OH(.).

Determination of penicillin in pharmaceutical formulations by flow injection analysis using an optimised immobilised penicillinase reactor and iodometric detection.

An automated assay for the determination of penicillin in formulations suitable for use in pharmaceutical quality control is presented. The method is based on the classical iodometric penicillin assay which is incorporated in a flow injection analysis (FIA) system. The required hydrolysis is performed on-line by using an immobilised penicillinase reactor. Packed-bed and single-bead-string enzyme reactors are compared. It turns out that a packed-bed penicillinase reactor (10 cm x 1.5 mm i.d.) provides complete hydrolysis within short residence time, while only little back-pressure is generated. This enzyme reactor is stable for at least 9 months. Enzymatic hydrolysis of the beta-lactam ring results in the formation of the corresponding penicilloic acid, which consumes iodine. The iodine consumption is determined colorimetrically by measuring the decrease of the absorbance of the blue coloured iodine/starch complex. The optimum reactor length and flow rate for the colourimetrical detection reaction are determined. The optimised method is applied to the assay of penicillin in formulations and the results are compared with the "true" results obtained with a reference method: a mercurimetric titration. The reliability of the flow injection method is evaluated quantitatively by determining the maximum total error (MTE). The reliability is shown to be highest when measuring at a 0.3-mM level. Eight formulations including capsules, tablets and injectables containing penicillin G, amoxicillin or flucloxacillin are assayed. The MTE does not exceed the 6% level and the most probable MTE is between 1.5 and 3.5%.

Determination of amikacin in serum by high-performance liquid chromatography with ultraviolet detection.

A procedure for the determination of amikacin in serum is described. The aminoglycoside is extracted from serum by using a disposable cation-exchange column. The eluate of this column is derivatized with 1-fluoro-2,4-dinitrobenzene and subsequently analysed by reversed-phase high-performance liquid chromatography with ultraviolet detection at 365 nm. The absolute recovery of amikacin by this procedure is 72%. Kanamycin is used as the internal standard. The sensitivity is 1 mg/l for amikacin with samples of 200 microliters. Precision, expressed as the coefficient of variation, is about 3% in the therapeutic concentration range. The 2,4-dinitrophenyl derivative of amikacin is synthesized on a preparative scale by a new method and its structure is demonstrated to be the fully derivatized amikacin. The analysis of serum samples obtained in an in vivo experiment correlates well with the results from a microbiological assay.