, Melissa Yssel, MB ChB, FC Path(SA) Chem 139, and Wendy M. Zakowicz, BS 79 Purpose: To achieve clinical validation of cutoff values for newborn screening by tandem mass spectrometry through a worldwide collaborative effort. Methods: Cumulative percentiles of amino acids and acylcarnitines in dried blood spots of approximately 25-30 million normal newborns and 10,742 deidentified true positive cases are compared to assign clinical significance, which is achieved when the median of a disorder range is, and usually markedly outside, either the 99th or the 1st percentile of the normal population. The cutoff target ranges of analytes and ratios are then defined as the interval between selected percentiles of the two populations. When overlaps occur, adjustments are made to maximize sensitivity and specificity taking all available factors into consideration.

Diazepam inhibits different isoforms of the enzyme cyclic nucleotide phosphodiesterase and also potentiates the inotropic effect of endogenous catecholamines in rat heart. In the present study we have examined whether this late effect is the consequence of inhibition of a phosphodiesterase subtype or whether inhibition of several phosphodiesterase subtypes is involved. We compared the effect of diazepam with that of the selective inhibitors of phosphodiesterase1 (MIMX), phosphodiesterase2 (EHNA), phosphodiesterase3 (milrinone) and phosphodiesterase4 (rolipram) on the inotropic effect of noradrenaline in rat ventricle. Both rolipram or diazepam were equipotent and more effective than milrinone in potentiating the inotropic effect of noradrenaline whereas EHNA and MIMX had no effect. The results suggest that the diazepam induced potentiation of the contractile effect of noradrenaline is due principally to inhibition of phosphodiesterase4 isoenzyme activity.We recently demonstrated that diazepam produces an inotropic and biochemical potentiation of endogenous catecholamines, namely noradrenaline and adrenaline (both exogenously applied or released by the indirectly acting sympathomimetic agent tyramine), in rat myocardium (Marín & Hernández 2002). This effect is not due to an enhancement of noradrenaline release at the presynaptic level nor is it mediated by b 2 -adrenoceptors or benzodiazepine receptors of the central or peripheral type (Marín & Hernández 2002). We also showed that diazepam exerts an inhibitory effect on different isoforms of the enzyme cyclic nucleotide phosphodiesterase (phosphodiesterase), including types 1, 2, 3 and 4 which are present in the heart and are involved in the hydrolysis of the cyclic AMP (cAMP) (Collado et al. 1998). This has functional relevance at a number of levels such as guinea-pig eosinophils (Collado et al.1998), rat aorta (Galindo et al. 2001 or the hypothalamicpituitary-adrenocortical axis of the rat . Furthermore, diazepam behaves like the non-selective phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine, in rat myocardium (Marín & Hernández 2002) and this phosphodiesterase inhibitory activity seems to mediate the diazepaminduced potentiation of the effects of certain inotropic agents acting via cAMP release, such as isoprenaline, the direct activator of adenylate cyclase, forskolin (Martinez et al. 1995) and histamine (Hara et al. 1998). Whether this effect is the consequence of inhibition of a particular phosphodiesterase subtype or whether a combined inhibitory action of diazepam on a number of phosphodiesterase subtypes is not Author for correspondence: J. Hernández; Department of Pharmacology; Faculty of Medicine; Campus de Espinardo; Murcia; Spain (fax π34 968 364150, e-mail jehernca/um.es).known. Thus the aim of the present study was to gain an insight into the respective roles of the four different phosphodiesterase subtypes in the diazepam induced potentiation of cAMP-dependent inotropic agents. To this end we compared the effect of diazepam wi...

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Glucagon increases cardiac contractility through G(s) protein-coupled glucagon receptors, but the inotropic responses fade. The fade could be due to receptor desensitisation or to the action of phosphodiesterases (PDE), or to both mechanisms. We investigated the effects of the PDE4 inhibitor rolipram (1 microM) on the inotropic and cAMP-responses to glucagon in paced right ventricular strips of the rat heart. Responses to the partial agonist dobutamine, mediated through beta(1)-adrenoceptors, were studied for comparison. Glucagon increased contractility (-logEC(50)M=7.3 for maximum responses with E(max)=32% of the response to 9 mM Ca(2+)), but the responses tended to fade (-logEC(50)M=7.1 for faded responses with E(max)=11.5%). Dobutamine (-logEC(50)M=5.8, E(max)=56%) produced positive inotropic effects that did not fade. Rolipram did not affect basal contractility and cAMP levels. Rolipram enhanced the contractile responses to glucagon and reduced fade (-logEC(50)M=7.5 and 7.3 with E(max)=74% and 45% for maximum and faded responses respectively). The response to glucagon (0.1 microM) completely faded in the absence of rolipram, but only partially faded and then remained stable in the presence of rolipram (1 microM). Rolipram enhanced contractile responses to dobutamine (-logEC(50)M=6.0, E(max)=75%). Dobutamine (3 microM), but not glucagon (0.1 microM), increased tissue levels of cAMP. Consistent with the inotropic data, rolipram caused glucagon to augment cAMP and enhanced the effects of dobutamine. Thus, PDE4 activity limits the responses mediated through both glucagon receptors and beta(1)-adrenoceptors. PDE4-catalysed hydrolysis of cAMP contributes to the inotropic tachyphylaxis of glucagon.

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Diazepam inhibits phosphodiesterase type 4 and enhances the effect of some 3',5'-cyclic adenosine monophosphate (cAMP)-dependent positive inotropic drugs. We sought to determine whether diazepam and the selective phosphodiesterase type 4 inhibitor rolipram enhances the contractile response and cAMP levels induced by dopamine in rat myocardium. Dopamine (3-100 microM) produced concentration-dependent positive inotropic effects (-log EC50 = 5.21 +/- 0.2, n = 5), which were augmented in the presence of 10 microM diazepam (-log EC50 = 5.40 +/- 0.08, n = 6, P < 0.05) or 1 microM rolipram (-log EC50 = 5.41 +/- 0.1, n = 6, P < 0.05). The effect of diazepam was not mimicked by 100 microM gamma-aminobutyric acid nor it was antagonized by a 5 microM concentration of the blockers of central and peripheral type benzodiazepine receptors, flumazenil and PK 11195. cAMP levels (pmol/g) produced by dopamine (744.4 +/- 111.8, n = 5) in this tissue were enhanced by the presence of diazepam (1073 +/- 97.7, n = 6, P < 0.05) or rolipram (1034.0 +/- 245.2, n = 5, P < 0.05). Therefore, diazepam, like rolipram, augments the inotropic and biochemical effects of dopamine in rat myocardium. This effect is not mediated by benzodiazepine receptors but is probably the consequence of the phosphodiesterase type 4 inhibitory activity of diazepam.

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