Assay of zofenopril and its active metabolite zofenoprilat by liquid chromatography coupled with tandem mass spectrometry

Zofenopril is a pro-drug designed to undergo metabolic hydrolysis yielding the active free sulfhydryl compound zofenoprilat, which is an angiotensin converting enzyme (ACE) inhibitor, endowed also with a marked cardioprotective activity. A simple, highly sensitive specific LC–MS–MS method was developed for the determination of zofenopril and zofenoprilat in human plasma. In order to prevent oxidative degradation of zofenoprilat and its internal standard, their free sulfhydryl groups were protected by treatment with N-ethylmaleimide (NEM), which produced the succinimide derivatives. The compounds and their corresponding fluorine derivatives, used as internal standards, were extracted from plasma with toluene. The reconstituted dried extracts were chromatographed and then monitored by a triple-stage-quadrupole instrument operating in the negative ion spray ionization mode. The method was validated over the concentration range of 1–300 ng/ml for zofenopril and 2–600 ng/ml for zofenoprilat. Inter- and intra-assay precision and accuracy of both zofenopril and zofenoprilat were better than 10%. The limit of quantitation was 1 ng/ml with zofenopril and 2 ng/ml with zofenoprilat. Extraction recovery proved to be on average 84.8% with zofenopril and 70.1% with zofenoprilat. Similar recoveries were shown by the above two internal standards. The method was applied to measure plasma concentrations of zofenopril and zofenoprilat in 18 healthy volunteers treated orally with zofenopril calcium salt at the dose of 60 mg.     

Release of diamine oxidase into plasma by glycosaminoglycans in rats

Plasma diamine oxidase (DAO) values are enhanced by intravenous injection of heparin which releases the enzyme, synthesized in small bowel enterocytes, from binding sites located on endothelial cells of the intestinal microvasculature. Intestinal DAO, in analogy with lipoprotein lipase (another heparin-released enzyme), is believed to be electrostatically linked to endothelial binding sites composed of a glycosaminoglycan (GAG) which is presumably heparan sulphate, but the complete mechanism of enzyme release is not known. In this study we assayed in rats the DAO-releasing capability of heparan sulphate, dermatan sulphate, chondroitin sulphate A and hyaluronic acid, all heparin related compounds. Heparan sulphate, a compound with the same hexosamine as heparin but with a lower concentration of sulphated iduronic acid, induced a very high release of DAO (3-fold less than heparin), while the other tested GAGs, composed of higher proportions of non sulphated uronic acid and with galactosamine instead of glucosamine, induced a significantly lower release. In rats treated with 60 mg heparan sulphate the significant decrease in ileal mucosal DAO activity indicates that, in analogy with heparin, the high plasma enzymatic activity induced is of enterocytic origin. It is suggested that the high charge density of the compounds tested, due to the degree of sulphatation, is the decisive factor in promoting the release of intestinal DAO.     

Diffusione pollinica e caratteristiche immunologiche e broncoreattive di soggetti atopici in Pietra Ligure (Savona)

Riassunto Sono state rilevate durante un monitoraggio aerobiologico effettuato a Pietra Ligure (Savona) nel corso del 1987 le concentrazioni polliniche di 50 taxa ed è stata valutata l'influenza relativa dei fattori meteorologici. Le osservazioni palinologiche sono state rapportate alle concentrazioni sieriche delle IgE specifiche, alla reattività bronchiale specifica ed aspecifica valutate in 101 pazienti allergici (rinitici ed asmatici), sensibilizzati a Graminaceae ed Urticaceae (Parietaria) al fine di riconoscere correlazioni tra le caratteristiche aerobiologiche di questi allergeni edi meccanismi patogenetici che sostengono la reattività bronchiale.      

Incidence of sensitisation to the pollens of urticaceae (Parietaria), Poaceae and Oleaceae (Olea europea) and pollen rain in Liguria (Italy)

Summary The authors examined 734 sensitised patients living in four localities in Liguria (Genoa, Savona, Pietra Ligure and Sanremo). The commonest source of sensitisation (62.7%) was Urticaceae (Parietaria), followed by Poaceae (52.5%) andOlea europaea L. (24.0%). A survey of airborne pollens revealed a greater presence of Urticaceae and Poaceae in Genoa and of Oleaceae in Pietra Ligure and Sanremo.     

Single amino acid substitutions in the reactive site of antithrombin leading to thrombosis. Congenital substitution of arginine 393 to cysteine in antithrombin Northwick Park and to histidine in antithrombin Glasgow

Antithrombin Northwick Park and antithrombin Glasgow are functionally variant antithrombins with impaired abilities to interact with thrombin. Thrombosis is associated with their inheritance. Both of the purified, reduced, and S-carboxymethylated variant antithrombins were treated with cyanogen bromide and the major pools of each containing the amino acid sequence Gly339-Met423 were isolated. Following treatment of these pools with trypsin, fast atom bombardment mass spectrometry identified tryptic peptides (found also in normal antithrombin treated in the same way) that corresponded to amino acid sequences Gly339-Lys370 and Val400-Met423. The tryptic peptides, corresponding to amino acid sequences Ala371-Arg393 and Ser394-Arg399 were present in both variant preparations in greatly reduced amounts compared to a normal antithrombin preparation. However, two novel tryptic peptides of molecular mass (M + H)+ 2976 and 2952 were identified in the digests of antithrombin Northwick Park and Glasgow, respectively. Further analyses of these novel tryptic peptides were carried out by V8 protease treatment and sequential Edman degradation coupled with mass spectrometric analysis of the shortened peptides. This established that these peptides comprised the amino acid sequence Ala371-Arg399, but with single amino acid substitutions at the reactive site, Arg393 replaced by Cys (in antithrombin Northwick Park) and by His (in antithrombin Glasgow).     

Amino Acid Analysis Demonstrates that Increased Plasma Free Tryptophan Causes the Increase of Brain Tryptophan During Exercise in the Rat

Rats were trained to run on a horizontal treadmill for 2 h at 20 m/min. This activity considerably increased plasma free tryptophan (TRP) (+70%) but did not alter plasma total TRP levels and had little or no effect on plasma concentrations of the other large neutral amino acids (LNAAs) that compete with TRP for entry into the brain. Brain TRP levels increased by 80%. The only other brain LNAA to be affected by exercise was threonine, which rose moderately. The results indicate that increased plasma free TRP was specifically responsible for the increase of brain TRP after 2 h of exercise. Brain lysine was also increased whereas glycine, alanine, and gamma-aminobutyric acid were decreased. The differences between the present findings and those previously obtained following 2 h immobilization stress are discussed.