Reliability of salivary theophylline in monitoring the treatment for apnoea of prematurity.

OBJECTIVE: To evaluate the reliability of salivary levels of theophylline in monitoring therapy of apnoea of prematurity. STUDY DESIGN: Aminophylline was administered intravenously in 13 infants with apnoea, in a loading dose of 5 mg/kg and maintenance dose of 3 mg/kg, every 8 h. The patients were divided into two groups according to their postconceptional age (PCA): group A, of infants with small PCA (32.8+/-2.0 weeks; n=6 cases), and group B, infants with higher PCA (37.1+/-0.8 weeks; n=7 cases). RESULTS: A total of 57 paired samples of serum and saliva were obtained in all 13 infants. The mean serum level of theophylline was 7.8+/-5.8 microg/ml and the ratio between serum and salivary concentration of theophylline was 1.53+/-0.28. A strong correlation between the serum and salivary concentration of theophylline (r=0.973) was found. Infants with small PCA had significant higher serum concentration of theophylline than those with higher PCA (10.6 vs 5.3 microg/ml; P=0.0002). The difference between the mean ratios of serum/salivary theophylline levels in the two groups was low (1.44 vs 1.62; P=0.0155). CONCLUSION: The strong correlation of theophylline in serum and in saliva recommends the salivary levels as a reliable method for monitoring the treatment of apnoea of prematurity.

Electron impact mass spectral interpretation for some thiophosphoryl-p-acetylaminobenzenesulfonamides.

This work discusses the synthesis and the fragmentation patterns for 2-(p-acetylaminosulfonamido)-2-thiono-(5,5-dimethyl-1,3,2-dioxaphosphorinane)(1) and for the p-acetylaminosulfonylamides of O,O-diethylthiophosphoric acid (2), O,O-diphenylthiophosphoric acid (3), dimethylaminocyclohexylthiophosphoric acid (4), and diethylaminophenylthiophosphoric acid (5). A thionamidic-thiolimidic structure was attributed to compounds 1-5, consistent with their IR and NMR spectra. EI mass spectra at 70 eV, high resolution (HR) mass measurements and metastable ion spectra were used to elucidate the fragmentation processes and to determine the kinetic energy release values associated with the metastable ion dissociations. HR accurate mass measurements were used to confirm the compositions of the more abundant ions.

[The monitoring of delayed-release theophylline therapy in bronchial asthma in children by determining the salivary concentration]. / Monitorizarea terapiei cu teofilina cu eliberare întârziata în astmul bronsic la copil prin determinarea concentratiei salivare.

Long-term therapy with slow-release theophylline is compulsory to be monitorized by measuring serum concentration of the drug. This is due to the narrow therapeutically index of theophylline for monitoring the long term therapy with this drug, and to establish the correlation between the serum and saliva concentration of theophylline. The study group was represented by 13 children with asthma, with the age between 2 and 13 years that received theophylline in an average dosage of 15.2 mg/kg/24 hours. In all of them there were obtained two paired simultaneous samples of venous blood and of saliva, at 4 hours and at 8 hours respectively, after the administration of theophylline. For facilitate the saliva sampling, its production was stimulated by applying citric acid on the tongue and aspiration with a sterile syringe. For measuring the serum and saliva levels of the drug it was used mass spectrometry with 15N-theophylline as internal standard. Serum theophylline concentration varied between 6.56 +/- 4.48 micrograms/ml at 8 hours after drug administration and respectively 7.45 +/- 3.94 micrograms/ml at 4 hours after drug administration. Saliva concentrations of theophylline varied between 4.18 +/- 2.40 micrograms/ml at 8 hours after drug administration and respectively 4.66 +/- 2.39 micrograms/ml at 4 hours after drug administration. The average ratio between serum and saliva concentration in the 24 paired samples was 1.62 +/- 0.18. The intraindividual variation of this ratio was 8.64%.

[The evaluation parameters of the long-term treatment efficacy of theophylline in the child with bronchial asthma]. / Parametrii de evaluare a eficientei tratamentului de durata cu teofilina la copilul cu astm bronsic.

In the past decade the use of theophylline was less extensive because of its narrow therapeutically index and due to its side effects. The widening of possibilities of monitoring the treatment by serum level determination, the introduction of slow release formula, and the demonstration of antiinflammatory effects made theophylline to become actual again. Theophylline is indicated for the chronic treatment of asthma in association with other medication. The authors study the efficacy and serum level of theophylline in 13 children with asthma who received slow release theophylline in a dosage of 15.2 mg/body weight/24 hours in average, administered 3 times a day. The efficacy was estimated by a clinical score and the serum level was determined by isotopic mass spectrometry with 15N-theophylline as internal standard. After a period of at least 2 months of therapy it was recorded the amelioration of clinical score (with 5.09 points, that represents 36.3% of the maximum initial score) and the reducing of circadian variation of peak expiratory flow from 27% to 15%. The serum level of theophylline was of 8.25 +/- 4.16 micrograms/ml at 4 hours after administration and of 5.69 +/- 2.6 micrograms/ml at 8 hours after administration of the last dose. The individual values less than 5 micrograms/ml was found in 3 of 13 children at 4 hours and in 7 of 13 children at 8 hours after the last dose. There were no patients with toxic serum levels of theophylline (> 20 micrograms/ml). The correlation between serum level of theophylline and the dosage was weak both at 4 hours (r = 0.054) and 8 hours (r = 0.229) after the last dose. At the same dose there were found high interindividual variations. These findings are arguments for the usefulness of determination of serum levels of theophylline, that together with clinical and functional parameters allowed the individualization of the dosage.

Trace analysis of triazines and organophosphorus pesticides in water.

A method for the determination of trace levels of triazines and organophosphorus pesticides in water is presented. The extraction method is based on a solid-phase extraction on C-18 bound silica SPE cartridges. A precolumn filled with Merck C-18 bound silica and home-made C-18 bound silica have been tested at a flow-rate of 3 ml/min with comparable preconcentration yields. A SIM-MS method using a (15)N labelled internal standard has been developed for the organophosphorus pesticides. Detection limits lower than 1 microg/L have been obtained. Separations have been carried out on a conventional GC column OV 17 (1 m) and a capillary column OV 17 (25 m) with a temperature program from 150 degrees C (2 min) to 300 degrees C (rate of 6 degrees C/min).

CMP kinase from Escherichia coli is structurally related to other nucleoside monophosphate kinases.

CMP kinase from Escherichia coli is a monomeric protein of 225 amino acid residues. The protein exhibits little overall sequence similarities with other known NMP kinases. However, residues involved in binding of substrates and/or in catalysis were found conserved, and sequence comparison suggested conservation of the global fold found in adenylate kinases or in several CMP/UMP kinases. The enzyme was purified to homogeneity, crystallized, and analyzed for its structural and catalytic properties. The crystals belong to the hexagonal space group P6(3), have unit cell parameters a = b = 82.3 A and c = 60.7 A, and diffract x-rays to a 1.9 A resolution. The bacterial enzyme exhibits a fluorescence emission spectrum with maximum at 328 nm upon excitation at 295 nm, which suggests that the single tryptophan residue (Trp30) is located in a hydrophobic environment. Substrate specificity studies showed that CMP kinase from E. coli is active with ATP, dATP, or GTP as donors and with CMP, dCMP, and arabinofuranosyl-CMP as acceptors. This is in contrast with CMP/UMP kinase from Dictyostelium discoideum, an enzyme active on CMP or UMP but much less active on the corresponding deoxynucleotides. Binding of CMP enhanced the affinity of E. coli CMP kinase for ATP or ADP, a particularity never described in this family of proteins that might explain inhibition of enzyme activity by excess of nucleoside monophosphate.

Chemienzymatic synthesis of uridine nucleotides labeled with [15N] and [13C].

UTP, labeled with 15N and 13C (at all carbon atoms of the ribose moiety), was obtained enzymatically from [15N]uracil and [13C6]glucose. Eleven enzymes and suitable substrates reconstituted a metabolic pathway in which glucose was first transformed to 5-phosphoribosyl-1-pyrophosphate. The latter compound plus uracil yielded UMP in a second step by the reaction catalyzed by uracil phosphoribosyltransferase. UMP was subsequently phosphorylated to the corresponding di- and triphosphate. ATP, required for five phosphorylation reactions, was regenerated from creatine phosphate, whereas NADP+ necessary for the oxidation of glucose 6-phosphate and 6-phosphogluconate was recycled by glutamate dehydrogenase and excess of ammonia and alpha-oxoglutarate. Despite the number and complexity of the enzymatic steps, the synthesis of [15N, 13C]UTP is straightforward with an overall yield exceeding 60%. This method, extended and diversified to the synthesis of all natural ribonucleotides, is a more economical alternative for obtaining nucleic acids for structural analysis by heteronuclear NMR spectroscopy.

Structural characterization by nuclear magnetic resonance spectroscopy of a genetically engineered high-affinity calmodulin-binding peptide derived from Bordetella pertussis adenylate cyclase.

This paper reports the solution conformation of a peptide (P196-267) derived from the calmodulin-binding domain of Bordetella pertussis adenylate cyclase. P196-267 corresponding to the protein fragment situated between amino acid residues 196-267 was overproduced by a recombinant Escherichia coli strain. Its affinity for calmodulin is only one order of magnitude lower (Kd = 2.4 nM) than that of the whole bacterial enzyme (Kd = 0.2 nM). The proton resonances of the NMR spectra of P196-267 were assigned using homonuclear two-dimensional techniques (double-quantum-filtered J-correlated spectroscopy, total correlation spectroscopy, and nuclear Overhauser enhancement spectroscopy) and a standard assignment procedure. Analysis of the nuclear Overhauser effect connectivities and the secondary shift distribution of C alpha protons along the sequence allowed us to identify the elements of regular secondary structure. The peptide is flexible in solution, being in equilibrium between random coil and helical structures. Two segments of 11 amino acids (situated between V215 and A225) and 15 amino acids (situated between L233 and A247) populate in a significant proportion the helix conformational state. The two helices can be considerably stabilized in a mixed solvent, trifluoroethanol/water (30/70), suggesting that the corresponding fragment in the intact protein assumes a similar secondary conformation. No elements of tertiary structure organization were detected by the present experiments. The conformational properties of the isolated calmodulin target fragment are discussed in relation with the available NMR and X-ray data on various peptides complexed to calmodulin.

Enzymatic synthesis of guanine nucleotides labeled with 15N at the 2-amino group of the purine ring.

GMP and dGMP labeled with 15N at the 2-amino group of the purine ring was obtained enzymatically from NH4Cl (> 99 at.% 15N) and from IMP or dIMP, respectively, by several reactions involving IMP-dehydrogenase, GMP-synthetase, adenylate kinase, and creatine kinase. The first three enzymes were obtained by overexpression in Escherichia coli of the corresponding genes. The isotope content of the primary amino group of guanine determined by mass spectrometry after acid hydrolysis of nucleotides was found higher than 98 at.% 15N. The proton NMR spectrum of [15N]GMP in solution in the absence of nitrogen decoupling showed a doublet with a coupling constant of 92 Hz. When nitrogen decoupling was used during the acquisition time, the doublet was replaced by a single peak at 6.47 ppm, indicating that the corresponding proton is bound to 15N.

Isotope dilution mass spectrometry for procaine determination in biological samples.

A simple, rapid and sensitive method for procaine determination is described. Isotope dilution mass spectrometry with (15N)procaine as internal standard was used. The analysis was performed at 4000 resolution by selected ion monitoring with temperature programming. The sample was measured in underivatized form in the direct inlet system. The method shows good analytical parameters: linearity between 0 and 40 micrograms ml-1, good precision and accuracy. The method was applied to the in vitro pharmacokinetic study of the metabolism of procaine in liver homogenates of Wistar rats. The method is rapid, permitting about six samples to be run per hour. Sensitivity of the method permits analysis at a signal-to-noise ratio of 5:1.