Manuel N.M.P. Alçada

Characterization of rat heart alkaline phosphatase isoenzymes and modulation of activity

Alkaline phosphatase (ALP) is important in calcification and its expression seems to be associated with the inflammatory process. We investigated the in vitro acute effects of compounds used for the prevention or treatment of cardiovascular diseases on total ALP activity from male Wistar rat heart homogenate. ALP activity was determined by quantifying, at 410 nm, the p-nitrophenol released from p-nitrophenylphosphate (substrate in Tris buffer, pH 10.4). Using specific inhibitors of ALP activity and the reverse transcription-polymerase chain reaction, we showed that the rat heart had high ALP activity (31.73 +/- 3.43 nmol p-nitrophenol.mg protein-1.min-1): mainly tissue-nonspecific ALP but also tissue-specific intestinal ALP type II. Both ALP isoenzymes presented myocardial localization (striated pattern) by immunofluorescence. ALP was inhibited a) strongly by 0.5 mM levamisole, 2 mM theophylline and 2 mM aspirin (91, 77 and 84%, respectively) and b) less strongly by 2 mM L-phenylalanine, 100 mL polyphenol-rich beverages and 0.5 mM progesterone (24, 21 to 29 and 11%, respectively). beta-estradiol and caffeine (0.5 and 2 mM) had no effect; 0.5 mM simvastatin and 2 mM atenolol activated ALP (32 and 36%, respectively). Propranolol (2 mM) tended to activate ALP activity and corticosterone activated (18%) and inhibited (13%) (0.5 and 2 mM, respectively). We report, for the first time, that the rat heart expresses intestinal ALP type II and has high total ALP activity. ALP activity was inhibited by compounds used in the prevention of cardiovascular pathology. ALP manipulation in vivo may constitute an additional target for intervention in cardiovascular diseases.

PVC membrane electrode without inner reference solution for the direct determination of ephedrine in pharmaceutical preparations

A PVC membrane electrode, without inner reference solution, based on an ion association extraction system responding to ephedrine is described. It incorporates an ephedrine-tetrakis (4-chlorophenyl)borate ion-pair complex in 2-nitrophenyloctyl ether. The prepared electrode exhibits a near Nernstian response (57.5 mV per decade) over the concentration range of 2 x 10(-5)-10(-1) M ephedrine in solutions of pH 2.5-9. The reproducibility of the electrode potentials were +/- 1 mV by day during at least 6 months. Response time was about 6 s for ephedrine concentrations between 10(-5) and 10(-1) M. Determinations of ephedrine in pharmaceutical preparations (tablets, nasal drops and syrups) by direct potentiometry gave an average recovery of 99.1% (w/w) and a mean standard deviation of 1.9% (w/w).

Quinidine ion-selective electrode for potentiometric determinations in pharmaceutical preparations

Abstract The construction and evaluation of a quinidine ion-selective electrode, without an inner reference solution, based on an ion exchanger [quinidine tetrakis(4-chlorophenycol)borate] dissolved in 2-nitrophenyl octyl ether and immobilized in PVC is described. The prepared electrode showed a linear response in the concentration range 3 × 10−5-1 × 10−2 M in quinidine, with a slope of 57.5 mV per decade when the ph of the solutions was between 5 and 7.5. The reproducibility of the electrode was approximately ±1 mV during a days work, with a response time of less than 10 s for quinidine concentrations between 10−4 and 10−2 M. The electrode was used for the direct potentiometric determination of quinidine in a pharmaceutical preparations, giving results with an average recovery of 100.7% and a mean standard deviation of 2.3%

FIA titrations of ephedrine in pharmaceutical formulations with a PVC tetraphenylborate tubular electrode.

A flow injection system for the titration of ephedrine in pharmaceutical products with potentiometric detection was developed. For this purpose a tetraphenylborate tubular electrode was constructed. The electrode was prepared without inner reference solution and with a PVC membrane based on tetrapentylammonium tetraphenylborate as ion exchanger and 2-nitrophenylphenyl ether as mediator solvent. Its operational characteristics were evaluated in a low dispersion manifold and compared with more conventionally shaped electrodes using the same sensor. In the pH range 2.5-11.5, the electrodes showed linear response between 3.8 x 10(-6) and 0.1 M with a slope of -56.4 mV/log[BPh4]. Ephedrine determinations in pharmaceutical products were carried out in a single channel manifold with a mixing chamber incorporated and using the tubular electrode as detector. Recovery rates of 98.6 +/- 2.5% were obtained in the analysis of tables, nasal drops and syrups with a sampling rate of about 60 h-1.

Application of poly(vinyl chloride) pilocarpine membrane electrodes in ophthalmic products

The construction and evaluation of two types of electrodes sensitive to pilocarpine, without an inner reference solution, based on pilocarpine–tetrakis(4-chlorophenyl) borate (Type I) and pilocarpine–tetrakis[3,5-bis(trifluoromethyl)-phenyl] borate (Type II) are described. The ion exchangers were dissolved in 2-nitrophenyl octyl ether and the resulting sensor solution was immobilized in poly(vinyl chloride)(PVC). Under similar experimental conditions, the calibration graphs for electrodes of Types I and II were linear in the concentration ranges between 3.0 × 10–5 and 6.0 × 10–5 to 0.1 mol dm–3(I= 0.1 mol dm–3 and pH = 6.3), respectively. The slopes of the calibration lines were close to the theoretical value and indistinguishable from each other. The reproducibilities of the potential for the two types of electrodes were similar, and approximately ±2 mV throughout a working day. The response time was better than 10 s for pilocarpine concentrations between 10–4 and 10–2 mol dm–3 for both types of electrodes. Both types of electrodes were in operation for a period exceeding 7 months. The electrodes exhibited useful analytical characteristics for the determination of pilocarpine in pharmaceutical products. Direct potentiometric calculations in solutions with a concentration of pilocarpine hydrochloride of about 48 µg cm–3 showed mean recoveries between 106 and 98% for determinations carried out with Type I electrodes and between 93 and 101% with Type II electrodes.