Correlated/non-correlated ion dynamics of charge-neutral ion couples: the origin of ionicity in ionic liquids.

Proton/fluoride spin-lattice (T1) nuclear magnetic relaxation dispersion (NMRD) measurements of 1-butyl-3-methyl-1H-imidazolium hexafluorophosphate, [C4mim][PF6], have been carried out using high field spectrometers and a fast-field-cycling instrument at proton Larmor frequencies ranging from 10 kHz to 40 MHz, at different temperatures. The NMRD profiles are interpreted by means of a simple relaxation model based on the inter- and intra-ionic dipole-dipole relaxation mechanism. Using an atomic molecular-ion dynamic simulation at 323 K the relevant spin dipole-dipole (DD) correlation functions are calculated. The results indicate that the NMRD profiles can be rationalized using intra- and inter-ionic spin DD interactions, however, anions are mainly modulated by ionic reorientation because of temporary correlations with cations, where modulation by translational diffusion plays a minor role. Reorientational dynamics of charge-neutral ion couples (i.e. [C4mim][PF6]) and [C4mim]+ ions are in the nano-second (ns) time range whereas the reorientation of [PF6]- is characterized by a reorientational correlation time in the pico-second (ps) regime. Based on the NMRD profiles we conclude that the main relaxation mechanism for [PF6]- is due to fast internal reorientational motion, a partially averaged F-F intra- and F-H inter-ionic DD coupling as the anion resides in close proximity to its temporary oppositely charged cation partner. The F-T1-NMRD data display a ns dispersion which is interpreted as being due to correlated reorientational modulations resulting from the H-containing charge-neutral ion couple [C4mim][PF6]. The analysis of ionicity is based on the free anion fraction, f, and it increases with temperature with f → 1 at the highest temperatures investigated. The fraction is obtained from the H-F NMRD profiles as correlated-non-correlated dynamics of the ions. The analysis of T1 relaxation rates of C, H, F and P at high fields cannot generally give the fraction of ions but is consistent with the interpretation based on the NMRD profiles with relaxation contributions due to DD-intra and -inter, CSA-intra (and -inter for C), including spin rotation for P. The investigation has led to a description of the mechanics governing ion transport in the title ionic liquid via identification of transient correlated/non-correlated ion dynamics.

Fluorescence spectroscopic properties analysed within the extended Förster theory with application to biomacromolecular systems.

The extended Förster theory (EFT) of electronic energy transport accounts for translational and rotational dynamics, which are neglected by the classical Förster theory (FT). EFT has been developed for electronic energy transfer within donor-acceptor pairs [Isaksson, et al, Phys. Chem. Chem. Phys., 9, 1941(2007)] and donor-donor pairs [Johansson, et al, J. Chem. Phys., 105, 10896 (1996); Norlin, et al, Phys. Chem. Chem. Phys., 10, 6962(2008)]. For donors that exhibit different or identical non-exponential fluorescence relaxation within a donor-donor pair, the process of reverberating energy migration is reversible to a higher or lower degree. Here the impact of the EFT has been studied with respect to its influence on fluorescence quantum yields, fluorescence lifetimes as well as depolarisation experiments. The FT predicts relative fluorescence quantum yields which usually agree with the EFT within experimental accuracy, however, substantial deviations occurs in the steady-state and in particular the time-resolved depolarisation data.

Albumin prevents metabolism of 12-hydroxyeicosatetraenoic acid by leukocytes in vitro.

In the present paper we studied the influence of albumin on the in vitro metabolism of 12-hydroxyeicosatetraenoic acid (12-HETE) and arachidonic acid in leukocytes and aspirin-treated platelets. In the presence of physiological concentrations of albumin, the metabolism of both 12-HETE and arachidonic acid was substantially altered, implicating the importance fatty acid binding proteins might have on the profile of products formed both in vitro and in vivo. The results clearly showed that albumin effectively withdraws arachidonic acid and 12-HETE from further metabolism by the leukocytes but does not influence the conversion of arachidonic acid to 12-HETE by the platelets. Thus, some of the hypotheses concerning transcellular metabolism raised from in vitro data within the eicosanoid field might have little relevance for the in vivo situation.

Evidence for a novel pathway of leukotriene formation in human platelets.

The metabolism of arachidonic acid via lipoxygenase-catalyzed reactions in washed human platelets was investigated. In addition to the previously discovered lipoxygenase metabolites, 12-hydroxyeicosatetraenoic acid, 15-hydroxyeicosatetraenoic acid, 8,15-dihydroxyeicosatetraenoic acid and 14,15-dihydroxyeicosatetraenoic acid, several other products were formed. The compounds were all dihydroxylated metabolites of arachidonic acid, containing a conjugated triene structure, and identified as 11,12-dihydroxyeicosatetraenoic acid (two isomers) and 5,12-dihydroxyeicosatetraenoic acid (four isomers). The identification was based on ultraviolet spectroscopy and gas chromatography-mass spectrometry of native and hydrogenated compounds. Stereochemical analysis of the hydroxyl groups of the 5,12-dihydroxyeicosatetraenoic acids and experiments with 18O2 indicated that the compounds were formed by the 12-lipoxygenase pathway, probably via an unstable epoxide.

Synthesis, structural identification and biological activity of 11,12-dihydroxyeicosatetraenoic acids formed in human platelets.

An enantiospecific route for the synthesis of 11,12-dihydroxyeicosatetraenoic acids was developed and used to synthesize 11,12-dihydroxy-5(Z),7(E),9(E),14(Z)-eicosatetraenoic acids. The 11,12-DHETEs were synthesized with the stereochemistry of the hydroxyl group being 11(R),12(S) and 11(S),12(S). The synthetic compounds were used to elucidate the structure of 11,12-DHETEs formed in human platelets by comparison of the chromatographic retention time in HPLC and GC as well as their ion fragmentation pattern in GC-MS. The major 11,12-DHETE formed in human platelets was found to be identical with 11(R),12(S)-dihydroxy-5(Z),7(E),9(E),14(Z)-eicosatetraenoic acid. Two more compounds were tentatively identified as 11(S),12(S)-dihydroxy-5(Z),7(E),9(E),14(Z)-eicosatetraenoic acid and 11,12-dihydroxy-5(E),7(E),9(E),14(Z)-eicosatetraenoic acid. Furthermore, the 11(S),12(S)-dihydroxy-5(Z),7(E),9(E),14(Z)-eicosatetraenoic acid was found to possess biological activity on neutrophil functional responses. However, the major compound, 11(R),12(S)-dihydroxy-5(Z),7(E),9(E),14(Z)-eicosatetraenoic acid, formed in platelets lacks biological activity in the test systems used. The present data further support that 11,12-dihydroxy-eicosatetraenoic acids are formed in human platelets via a leukotriene like mechanism presumably by the 12-lipoxygenase. Furthermore, the biological effects of one of the compounds showed a unique activity profile compared to other lipoxygenase products.

11-Hydroxythromboxane B2 dehydrogenase is identical to cytosolic aldehyde dehydrogenase.

11-Hydroxythromboxane B2 dehydrogenase purified from porcine kidney has been identified as cytosolic aldehyde dehydrogenase (EC 1.2.1.3). This identification is based on protein characteristics, sequence analysis of one proteolytic digest, blocked N-terminus, subunit molecular mass of 55 kDa, and enzymatic activities. The sequence difference with the human enzyme is 7.5% in the fragments analyzed (29 exchanges of 388 positions, corresponding to the expected species variability for cytosolic aldehyde dehydrogenase). The substrate thromboxane B2 contains a hemiacetal in its ring structure, but the reaction most likely proceeds via the aldehyde form of the substrate. This finding is in agreement with the proposed metabolism of 4-hydroxycyclophosphamide and highlights the possibility that molecules containing a hemiacetal structure can function as substrates for aldehyde dehydrogenase.

Paramagnetic proton nuclear spin relaxation theory of low-symmetry complexes for electron spin quantum number S = 52.

A generalization of the modified Solomon-Bloembergen-Morgan (MSBM) equations has been derived in order to describe paramagnetic relaxation enhancement (PRE) of paramagnetic complexes characterized by both a transient (DeltaZFSt) and a static (DeltaZFSs) zero-field splitting (ZFS) interaction. The new theory includes the effects of static ZFS, hyperfine coupling, and angular dependence and is presented for the case of electron spin quantum number S = 52, for example, Mn(II) and Fe(III) complexes. The model gives the difference from MSBM theory in terms of a correction term delta which is given in closed analytical form. The theory may be important in analyzing the PRE of proton spin-lattice relaxation dispersion measurements (NMRD profiles) of low-symmetry aqua-metal complexes which are likely to be formed upon transition metal ions associated with charged molecular surfaces of biomacromolecules. The theory has been implemented with a computer program which calculates solvent water proton T1 NMRD profiles using both MSBM and the new theory.

12-hydroxyeicosatetraenoic acid is a long-lived substance in the rabbit circulation.

12-Hydroxyeicosatetraenoic acid (12-HETE) is one of the major metabolites formed from arachidonic acid in platelets. We have recently shown that the in vitro metabolism of 12-HETE by human leukocytes, with and without stimulation, is effectively inhibited by the addition of physiological concentrations of albumin, probably by sequestration of the compound. In the present paper, we have studied the in vivo metabolism of 12-HETE in the rabbit, using either [1-14C]- or [14C(U)]12-HETE. Distribution of radioactivity was followed in urine, plasma, and bile, as well as in a number of tissues. In most of the tissues examined, the hydrophilic radioactivity constituted more than 50% of the total radioactivity after 20 min. When the lipophilic fraction was analyzed, around 15% of the radioactivity was shown to be unesterified 12-HETE, and only a very minor part could be detected as metabolites. The dominating lipophilic compound in the circulation after i.v. administration of radiolabeled 12-HETE was at all time points (1-60 min.) the parent compound, as analyzed by HPTLC and HPLC. A comparison of the plasma metabolite profiles obtained when [1-14C]- and [14C(U)]12-HETE were used displayed almost identical patterns, thus indicating that beta-oxidized metabolites either were not formed or were rapidly removed from the circulation. The appearance of large amounts of water-soluble radioactivity with time supported the latter conclusion. Several minor metabolites were seen that chromatographed in the dihydroxy acid region as judged by HPLC and TLC. The major one of these compounds represented about 10% of the lipophilic plasma radioactivity after 60 min., while unmetabolized 12-HETE at this stage still represented about 30%. The metabolite had a polarity similar to 12,20-dihydroxyeicosatetraenoic acid; however, when chromatographed together, these two compounds separated, indicating a different structure of the metabolite. Our findings are in agreement with in vitro data concerning the protective effect of albumin on the metabolism of 12-HETE and is the first extensive metabolic study of 12-HETE in vivo covering all metabolic possibilities involving the carbon skeleton.

Bioavailability of norethisterone acetate alone and in combination with estradiol administered in single or multiple oral doses to postmenopausal women.

OBJECTIVES: Twenty-four postmenopausal women were randomly allocated to a cross-over trial for an investigation of the pharmacokinetics of norethisterone acetate (NETA; 0.5 mg), administered alone or in combination with estradiol (E2; 1 mg), both after a single oral dose. In a second trial, the above combination of 0.5 mg NETA with 1 mg E2 was administered daily for 28 days. METHODS: Plasma levels of NET, E2, estrone (E1) and estrone sulphate fraction containing an admixture of estrone glucuronide (E1S/E1G) were measured by radioimmunoassay at various intervals up to 72 h in the first trial and at the same intervals after the 28th day in the second trial. RESULTS: In the first, single-dose trial, pharmacokinetic parameters of NET were similar for NETA administered alone and its combination with E2. There was no statistically significant difference in the area under curve values AUC0-24 and AUC0-infinity and no apparent major differences were observed for other pharmacokinetic parameters. No carry-over effects due to the cross-over design were seen. The multiple dosage in the second trial did not cause any major changes in the pharmacokinetic parameters of NET, except for the AUC0-24 and AUC0-infinity values which were significantly higher than those seen in the first trial. The levels of E2 exhibited, shortly after the intake of E2, a rapid burst. The levels gradually decreased to a nadir followed by an increase to the main peak and by the subsequent elimination phase. The difference between the peak and nadir levels was significant (P < 0.05) in the second, multiple-dose trial. This bimodal pattern was not observed in earlier studies. The main metabolite of E2 was E1S/E1G, followed by E1, as could be seen from the AUC0-infinity values. These were, in both trials, approximately 300 and 7-times higher for the E1S/E1G and E1, respectively, than those for E2. For all analytes, the AUC0-24 values were significantly higher in the second trial than those found in the first trial, indicating accumulation upon repeated administration. Pharmacokinetics of all analytes remained linear in the second trial, as follows from the statistically established equality of AUC0-24 found in the second, multiple-dose trial with AUC0-infinity in the first, single-dose trial. The absorption half-life and t-max values of E1S/E1G appeared to be considerably shorter than those of E1 in both trials. CONCLUSIONS: The bioavailability of NET was not influenced by its combination with 1 mg E2. The most abundant metabolite of E2 was the E1S/E1G fraction, which may have served as the main source of E2 and other estrogens due to metabolic interconversions during the absorption and elimination phases.

Extended Förster theory for determining intraprotein distances. Part III. Partial donor-donor energy migration among reorienting fluorophores.

An extended Förster theory (EFT) is derived and outlined for electronic energy migration between two fluorescent molecules which are chemically identical, but photophysically non-identical. These molecules exhibit identical absorption and fluorescence spectra, while their fluorescence lifetimes differ. The latter means that the excitation probability becomes irreversible. Unlike the case of equal lifetimes, which is often referred to as, donor-donor energy migration (DDEM), the observed fluorescence relaxation is then no longer invariant to the energy migration process. To distinguish, the present case is therefore referred to as partial donor-donor energy migration (PDDEM). The EFT of PPDEM is described by a stochastic master equation (SME), which has been derived from the stochastic Liouville equation (SLE) of motion. The SME accounts for the reorienting as well as the translational motions of the interacting chromophores. Synthetic fluorescence lifetime and depolarisation data that mimics time-correlated single photon counting experiments have been generated and re-analysed. The rates of reorientation, as well as the orientational configurations of the interacting D-groups were examined. Moreover the EFT of PPDEM overcomes the classical "kappa(2)-problem" and the frequently applied approximation of kappa(2) = 2/3 in the data analyses. An outline for the analyses of fluorescence lifetime and depolarisation data is also given, which might prove applicable to structural studies of D-labelled macromolecules, e.g. proteins. The EFT presented here brings the analyses of PDDEM data to the same level of molecular detail as that used in ESR- and NMR-spectroscopy.

Extensive NMRD studies of Ni(II) salt solutions in water and water-glycerol mixtures.

Aqueous solutions of simple nickel(II) salts are a classical test case for theories of the paramagnetic relaxation enhancement (PRE) and its dependence on the magnetic field (nuclear magnetic relaxation dispersion, NMRD), going back to late fifties. We present here new experimental data, extending the NMRD range up to 21T (900 MHz). In addition to salt solutions in (acidified) water, we have also measured on solutions containing glycerol. The aqueous solution data do not show any significant changes compared to the earlier experiments. The interpretation, based on the general ("slow-motion") theory is also similar to the earlier work from our laboratory. The NMRD-data in mixed solvents are qualitatively different, indicating that the glycerol not only changes the solution viscosity, but may also enter the first coordination sphere of the metal ion, resulting in lower symmetry complexes, characterized by non-vanishing averaged zero-field splitting. This hypothesis is corroborated by molecular dynamics simulations. A strategy appropriate for interpreting the NMRD-data for the chemically complicated systems of this type is proposed.

Purification and some properties of 11-hydroxythromboxane B2 dehydrogenase from porcine kidney.

A protein with NAD-dependent 11-hydroxythromboxane B2 dehydrogenase activity was purified to apparent homogeneity from porcine kidney using a relatively simple purification procedure, involving precipitation, anion-exchange chromatography (diethylaminoethyl-cellulose), affinity chromatography (5'-AMP-Sepharose) and gel-filtration chromatography (Protein Pak 125). The dehydrogenase was found to have a molecular mass of 50-55 kDa as determined by comparison with standards on SDS/PAGE. The molecular mass on gel-filtration chromatography was dependent on the ionic strength of the buffer. The apparent Km and Vmax values for thromboxane B2 were also dependent of the ionic strength with a Vmax of 214 nmol min-1 mg-1 using 250 mM Tris/HCl, pH 8.0, and a corresponding Km of 2.9 mM. The enzyme was NAD dependent and was clearly separated from the proteins with 15-hydroxyprostaglandin dehydrogenase activity also present in the kidney. Furthermore, it was found that 11-hydroxythromboxane B2 dehydrogenase did not utilize prostaglandin D2, prostaglandin E2, prostaglandin F2 alpha or cholic acid as substrate, and that the enzyme did not catalyse the reverse reaction, conversion of 11-dehydrothromboxane B2 to thromboxane B2.

Purification to homogeneity of an NAD dependent 11-hydroxythromboxane B2 dehydrogenase from porcine kidney.

A protein with NAD dependent 11-hydroxythromboxane B2 dehydrogenase activity was purified to homogeneity from porcine kidney using a simple purification procedure, involving precipitation, anion exchange chromatography (DE-52), affinity chromatography (5'-AMP-Sepharose) and gel filtration chromatography (Protein Pak 300SW). The dehydrogenase was found to have a molecular mass of 50 kDa and 42 kDa as determined by comparison with standards on SDS/PAGE and gel filtration chromatography respectively. The apparent Km and Vmax values for thromboxane B2 were 220-250 microM and 15-30 nmol min-1 mg-1 respectively. The enzyme was clearly separated from the proteins with 15-hydroxyprostaglandin dehydrogenase activity also present in the kidney. Furthermore it was found that 11-hydroxythromboxane B2 dehydrogenase did not utilize prostaglandin D2 prostaglandin E2 or prostaglandin F2 alpha as substrate, and that the enzyme did not catalyze the reverse reaction, conversion of 11-dehydrothromboxane B2 to thromboxane B2.

Albumin modifies the metabolism of hydroxyeicosatetraenoic acids via 12-lipoxygenase in human platelets.

12-Lipoxygenase and cyclooxygenase 1 are the dominating enzymes that metabolize arachidonic acid in human platelets. In addition to the conversion of arachidonic acid to 12(S)-hydroxyeicosatetraenoic acid, 12-lipoxygenase can also utilize 5(S)-hydroxyeicosatetraenoic acid and 15(S)-hydroxyeicosatetraenoic acid to form 5(S), 12(S)-dihydroxyeicosatetraenoic acid and 14(R), 15(S)-dihydroxyeicosatetraenoic acid, respectively. Furthermore, 15(S)-hydroxyeicosatetraenoic acid works as an inhibitor for 12-lipoxygenase. In the present paper we have studied the influence of albumin on the in vitro metabolism of 5 - and 15 -hydroxyeicosatetraenoic acids, and 5,15 -dihydroxyeicosatetraenoic acid by the platelet 12-lipoxygenase. The presence of albumin reduced the formation of 5(S),12(S)- dihydroxyeicosatetraenoic acid from 5(S)-hydroxyeicosatetraenoic acid, however, it had no effect on the 12(S)-hydroxyeicosatetraenoic acid production from endogenous arachidonic acid. In contrast, when 15(S)-hydroxyeicosatetraenoic acid was incubated with activated platelets, the formation of 14(R), 15(S)- dihydroxyeicosatetraenoic acid was stimulated by the presence of albumin. Furthermore, albumin reduced the inhibitory action 15(S)-hydroxyeicosatetraenoic acid had on 12(S)-hydroxyeicosatetraenoic acid formation from endogenous arachidonic acid. However, addition of exogenous arachidonic acid (20 microm) to the incubations inverted the effects of albumin on the conversion of 15(S)-hydroxyeicosatetraenoic acid to 14(R),15(S)- dihydroxyeicosatetraenoic acid and the production of 12(S)-hydroxyeicosatetraenoic acid in these incubations. Based on the Scatchard equation, the estimates of the binding constants to albumin were 1.8 x 10(5) for 15 -HETE, 1.4 x 10(5) for 12-HETE, and 0.9 x 10(5) for 5 -HETE respectively. These results suggest an important role of albumin for the regulation of the availability of substrates for platelet 12-lipoxygenase.

Theoretical nuclear magnetic resonance-lineshape study of a two-site chemical exchange model of sodium ions (I = 3/2) in an intracellular environment.

We present a theoretical calculation of the lineshape function based on the solution of the semiclassical Liouville equation, of a two-site chemical exchange model of biological relevance. The bound site is allowed to be in the slow region regime that is the inverse quadrupole interaction of one bound site is in the same range as the reorientational correlation time. We compare different chemical exchange models, and several different physical situations are investigated. The variation of the width at half height (WHH) and the relative intensity (l/lo) is shown to be important, experimentally accessible quantities that are useful in order to discriminate between different model systems.

Circulating and urinary thromboxane B2 metabolites in the rabbit: 11-dehydro-thromboxane B2 as parameter of thromboxane production.

The metabolism of thromboxane B2 was studied in the rabbit. The aim of the study was to identify metabolites in blood and urine that might serve as parameters for monitoring thromboxane production in vivo. [5,6,8,9,11,12,14,15-3H8]-Thromboxane B2 was administered by i.v. injection to rabbits, and blood samples and urine were collected with brief intervals. The metabolic profiles were visualized by two-dimensional thin layer chromatography and autoradiography, and the structures of five major metabolites were determined using chromatographic and mass spectrometric methods. In urine the major metabolites were identified as 11-dehydro-TXB2 and 2,3,4,5-tetranor-TXB1, and other prominent products were 11-dehydro-2,3,4,5-tetranor-TXB1, 2,3-dinor-TXB1 and 2,3-dinor-TXB2. In the circulation, TXB2 was found to disappear rapidly. The first major metabolite to appear was 11-dehydro-TXB2, which also remained a prominent product in blood for the remainder of the experiment (90 min). With time, the profile of circulating products became closely similar to that in urine. TXB2 was not converted into 11-dehydro-TXB2 by blood cells or plasma. The dehydrogenase catalyzing its formation was tissue bound and was found to have a widespread occurrence: the highest conversion was found in lung, kidney, stomach and liver. The results of the present study suggest that 11-dehydro-TXB2 may be a suitable parameter for monitoring thromboxane production in vivo in the rabbit in blood as well as urinary samples, and possibly also several tissues. This was also demonstrated in comparative studies using radioimmunoassays for TXB2 and 11-dehydro-TXB2.

Identification of 11-dehydro-TXB2 as a suitable parameter for monitoring thromboxane production in the human.

In order to identify suitable parameters for measurement of thromboxane production in vivo, the metabolism of TXB2 was studied in the human. [3H8]-TXB2 was given intravenously to a healthy human volunteer. Blood samples were collected for 50 min after the injection, and urine was collected for 24 hours. The urinary and blood metabolic profiles were visualized by the use of two-dimensional TLC and autoradiography. Identification of metabolites was achieved with GC/MS and in some cases by cochromatography with reference compounds in TLC and GC. In blood, unmetabolized TXB2 was the dominating compound during the first 30 min. Three less polar metabolites appeared, two of which were identified as 11-dehydro-TXB2 and 11,15-didehydro-13,14-dihydro-TXB2, respectively. The third compound was tentatively identified as 15-dehydro-13,14-dihydro-TXB2. Since 11-dehydro-TXB2 was one of the major metabolites in blood as well as urine, it was deemed suitable as target for measurement of thromboxane production in vivo. The advantages of 11-dehydro-TXB2 over its parent compound, TXB2, were demonstrated in experiments where unlabeled TXB2 was injected i.v. to a human volunteer, and the blood and urinary levels of both compounds were then followed by radioimmunoassay. Measured levels of 11-dehydro-TXB2 were found to give a more reliable picture of metabolic events than TXB2, the latter compound to a large extent reflecting technical difficulties during blood sample collection.