Kinetics of release of heparin from alginate hydrogel.

PURPOSE: Injected sodium alginate may be a useful perivascular drug delivery vehicle. This study was performed to determine the release rates of heparin from sodium alginate hydrogels cross-linked with varying amounts of calcium gluconate. MATERIALS AND METHODS: Six hydrogels, composed of 0.16 mEq sodium alginate and 4,000 units unfractionated heparin, were cross-linked with calcium gluconate to yield ion equivalence (IE) ratios (calcium:alginate) of 0.2, 0.4, 0.58, 0.8, 1.0, or 1.2. Two milliliters of normal saline was placed on top of each gel and allowed to remain in contact for up to 10 days. At set time intervals, the amount of heparin in the eluent was determined with use of high-performance liquid chromatography. RESULTS: Gels with 0.2 and 0.4 IE were partially liquid at 24 hours; the other gels solidified within 10 minutes. The 0.58 IE gel was slowest to solidify but immobilized the most heparin and released heparin slowest over 10 days. At 10 days, between 5.5% and 9.8% of the heparin immobilized was retained in the gel. CONCLUSION: This hydrogel shows promise as a vehicle for in vivo perivascular heparin delivery. The 0.58:1 IE ratio hydrogel has slowest release rate and the greatest immobilization despite its longer cross-linking time.

Conformation of manganese(II)-nucleotide complexes bound to rabbit muscle creatine kinase: 13C NMR measurements using [2-13C]ATP and [2-13C]ADP.

Conformations of cation-nucleotide complexes bound to rabbit muscle creatine kinase were investigated by measuring paramagnetic effects on 13C spin relaxation in E.Mn[2-13C]ATP and E.Mn[2-13C]ADP at three different frequencies, viz., 50, 75, and 125 MHz, and as a function of temperature in the range of 7-35 degrees C (at 75 MHz). Arrhenius plots of the temperature dependencies of relaxation rates show a positive slope with low activation energies of 1.3 +/- 0.2 kcal/mol and 2.0 +/- 0.2 kcal/mol for E.Mn ATP and E.MnADP, respectively. The relaxation rates of both complexes show strong frequency dependence, indicating that these rates are not exchange limited. Analysis of the data yields Mn(II)-2C distances of 10.0 +/- 0.5 A for E.MnATP and 8.6 +/- 0.5 A for E.MnADP. These data were interpreted, along with previously published information, on the location of the cation with respect to the phosphate chain [Jarori, G. K., Ray, B.D., & Nageswara Rao, B. D. (1985) Biochemistry 24, 3487-3494], and on the adenosine conformation [Murali, N., Jarori, G. K., & Nageswara Rao, B. D. (1993) Biochemistry 32, 12941-12948] in these complexes. The Mn(II)-2C distances depend on the orientation of the phosphate chain relative to the adenosine moiety. Conformational searches were performed by varying the two torsion angles, phi 1 (C4'-C5'-O5'-P alpha), and phi 2 (C5'-O5'-P alpha-O alpha beta), along with CHARMm energy computations, in order to determine acceptable conformations compatible with the distances determined. The significant difference in the Mn(II)-2C distances in E.MnATP and E.MnADP is indicative of the structural alterations occurring at the active site as the enzyme turns over.

Structural characterization and sequence distributions of polysiloxanes using pyrolysis MS/MS.

Novel polysiloxanes, with 4-(dialkylamino)pyridine substituents, are characterized by pyrolysis tandem mass spectrometry. These polymers form abundant cyclic oligomeric ions under both desorption electron ionization (DEI) and desorption chemical ionization (DCI) conditions. Product MS/MS spectra of the cyclic ions reveal characteristic fragmentations under low energy collision activated dissociation. Protonated cyclic oligomers higher than the pentamer are mainly due to the proton bound dimers of lower oligomeric units. The cyclic oligomers are shown to have proton affinities greater than 1000 kJ/mole. It is proposed that thermal depolymerization occurs through an intramolecular siloxane bond rearrangement, which is in agreement with a previously proposed "loop mechanism". Markovian statistical calculations are applied to the DCI mass spectral data in order to determine the sequence distribution of siloxane copolymers. Application of this method show that the monomers in the copolymers examined are non-randomly distributed.

Structural characterization of poly(N-alkyl-4-vinylpyridinium) triflates using pyrolysis/tandem mass spectrometry.

Desorption chemical ionization (DCI) and desorption electron ionization (DEI) of homo- and co-polymers of N-alkyl-4-vinylpyridinium triflates having ethyl, n-hexyl and n-dodecyl groups as N-alkyl substituents, produce mass spectra that display oligomeric ions. These positively charged ions are singly-charged and result from cleavage of the polymer into neutral oligomers and the loss of a single triflate anion per oligomer. Analogous negatively charged ions, in which each neutral oligomer carries an extra triflate anion, are observed in the desorption chemical ionization mass spectra. Each oligomer within the available mass range is represented in the mass spectra. The formation of cluster ions in which a single, multiply-charged cation is associated with a number of singly-charged anions, as observed for these ammonium polysalts, is unusual. Five major and three minor series of positively charged ions are observed in DCI and DEI methods of ionization. Ions in the different series correspond either to cleavage at different bonds between the constituent monomers or to hydrogen transfer in different directions. Unique and structurally diagnostic fragmentation processes are observed in tandem mass spectrometry (MS/MS) experiments performed using collision activated dissociation of mass-selected oligomeric ions.

Slow reversible inhibition of rabbit muscle aldolase by D-erythrulose 1-phosphate.

Rabbit muscle aldolase was found to be inactivated in a slow, reversible manner by D-erythrulose 1-phosphate. This compound combined rapidly and reversibly with the enzyme to form an initial complex, which then only slowly (ki = 0.28 min-1) converted to a kinetically more stable form. This stable enzyme-ligand form was inactive toward the normal substrate of aldolase, fructose 1,6-bisphosphate. The inactive enzyme-ligand complex, however, could be decomposed (kr = 0.0041 min-1) to yield active enzyme once again by incubation in a solution devoid of D-erythrulose 1-phosphate.

5,5-Bis(3-hydroxyphenyl)hydantoin, a minor metabolite of diphenylhydantoin (dilantin) in the rat and human.

5,5-Bis(4-hydroxyphenyl) hydantoin has been identified as a minor metabolite of diphenylhydantoin (DPH) in the rat and human. This metabolite was synthesized in the laboratory from 4,4'-dihydroxybenzophenone. Gas chromatographic and mass spectrometric comparison of the permethylated synthetic compound with the permethylated derivative of the metabolite obtained from biological sources showed that they were identical. The metabolite was excreted as a glucuronide and accounted for about 1% of the total hydroxylated metabolites of DPH in human urine and rat bile. When the synthetic standard was added to the recirculating perfusate of the isolated perfused rat liver, a monoglucuronide, a trihydroxy-DPH glucuronide, and a dihydroxymethoxy-DPH glucuronide were identified in the bile.