Competitive inhibition of delta8-tetrahydrocannabinol and its active metabolites for cannabinoid receptor binding

In vitro binding characteristics of delta8-tetrahydrocannabinol (delta8-THC) and its metabolites, 11-hydroxy-delta8-THC (11-OH-delta8-THC) and 11-oxo-delta8-THC, as well as an inactive metabolite, delta8-THC-11-oic acid, as a cannabinoid receptor site from bovine cortex were examined using the specific agonist [3H]CP-55940. 11-OH-delta8-THC and 11-oxo-delta8-THC strongly inhibited the specific binding of [3H]CP-55940. The Ki values of 11-OH-delta8-THC and 11-oxo-delta8-THC for the specific binding of [3H]CP-55940 were 52 and 143 nM, respectively, whereas that of delta8-THC-11-oic acid was 917 nM. Scatchard plot analyses indicated that 11-OH-delta8-THC and 11-oxo-delta8-THC caused a significant increase in the apparent KD value without changing the apparent Bmax. These results reveal that active metabolites of delta8-THC also competitively bind to the cannabinoid receptor as agonists.     

Saikosaponins from roots of Bupleurum scorzonerifolium

Saikosaponin u and saikosaponin v, were isolated from the roots of Bupleurum scorzonerifolium and these saponins were identified as 3-O-[beta-D-glucopyranosyl-(1-->2)-beta-D-glucopyranosyl- (1-->3)-beta-D-fucopyranosyl]-3 beta,16 alpha,23,28-tetrahydroxy-olean- 11,13(18)-dien-30-oic acid-30-O-[pentito(1-->1)-beta-D-glucopyranosyl-(6-->)] ester and 3-O-[beta-D-glucopyranosyl-(1-->3)-beta-D-fucopyranosyl]-3 beta,16 alpha,23,28-tetrahydroxy-olean-11,13(18)-dien-30-oic acid-30-O-[pentito(1-->1)-beta-D-glucopyranosyl(6-->)] ester, respectively.     

Developmental changes in rat diaphragm endplate response to repetitive stimulation

Three new triterpenoid saponins, their methyl esters designated as castaralesides F (1), G (2) and H (3), were isolated from the fresh leaves of Castanospermum australe. The structures of these three saponins were elucidated on the basis of chemical and spectral data as 3 beta-O-[beta-galactopyranosyl-(1-->4)-beta-glucuronopyranosyl]-2 beta,23-dihydroxyolean-12-en-28-oic acid; 3 beta-O-[alpha-rhamnopyranosyl-(1-->4)-beta-galactopyranosyl-(1-->2)-beta - glucurono-pyranosyl]-2 beta,28-dihydroxyolean-12-ene; and 3 beta-O-[alpha-rhamnopyranosyl-(1-->4)-beta-xylopyranosyl-(1-->2)-beta- glucuronopyranosyl]-2 beta,28-dihydroxyolean-12-ene.     

Selective in vivo binding of [3H]naltriben to delta-opioid receptors in mouse brain

Naltriben (NTB) is a selective antagonist for the putative delta2-opioid receptor. We have determined the regional kinetics and pharmacological profile of [3H]naltriben in vivo in mouse brain. After i.v. administration to CD1 mice, [3H]naltriben uptake and retention were high in striatum, cortical regions and olfactory tubercles, and low in superior colliculi and cerebellum. Robust rank order correlation was found between [3H]naltriben uptake in discrete brain regions and prior delta-opioid receptor binding determinations in vitro and in vivo. [3H]Naltriben binding in vivo was saturable, and was blocked by the delta-opioid receptor antagonist naltrindole, but not by the mu-opioid receptor antagonist cyprodime or the K-opioid receptor agonist (trans)-(+/-)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]ben zeneacetamide mesylate (U50,488H). (E)-7-Benzylidenenaltrexone (BNTX), a selective antagonist for the putative delta1-opioid receptor, was 9.6- to 12.9-fold less potent than naltriben as an inhibitor of [3H]naltriben binding. Thus, the sites labeled by [3H]naltriben in vivo may correspond to the delta2-opioid receptor subtype. Such assignment is not definitive, particularly considering the 4-fold higher brain uptake of naltriben as compared to (E)-7-benzylidenenaltrexone. Moreover, the regional distribution of [3H]naltriben in brains from CXB-7/BY (CXBK) mice, a strain that shows supraspinal delta1- but not delta2-opioid receptor agonist effects, was quite similar to that found for CD1 mice.     

Estimation of Koc values for deuterated benzene, toluene, and ethylbenzene, and application to ground water contamination studies

Sorption partition coefficients between water and organic carbon (Koc) for deuterated benzene, toluene, and ethylbenzene have been estimated by measuring values of the octanol-water partition coefficient (Kow) and HPLC retention factors (k1), which correlate closely to values of Koc. Measured values of log Kow for non-deuterated and deuterated toluene are 2.77 (+/- 0.02) and 2.78 (+/- 0.04), respectively, indicating that within experimental error, log Koc for deuterated and non-deuterated toluene are the same. The HPLC method provides greater precision, and yields values of delta log Koc (= log Koc [deuterated]-log Koc [non-deuterated]) of -0.021 (+/- 0.001) for benzene, -0.028 (+/- 0.002) for toluene, and -0.035 (+/- 0.003) for ethylbenzene. The small values of delta log Koc demonstrates that deuterated compounds are excellent tracers for the hydrologic behavior of ground water contaminants.     

Plasma transport forms of ingested fatty alcohols in the rat

Previous studies have shown that ingested fatty alcohols are absorbed as fatty acids and fatty acid esters, particularly triglycerides. The present study was carried out to determine whether fatty alcohols are also transported as 0-alkyl glyceryl ethers, alk-1-enyl glyceryl ethers, and as wax esters. Oxidation of fatty alcohols to other lipids was assessed by using a mixture of [1-3H] hexadecanol and [1-14C] hexadecanol of predetermined ratio. The results indicate that the absorption of fatty alcohol, and of its transport forms, parallels the absorption of labeled fatty acids. Six to 25% of plasma radioactivity was present as 1-0-alkyl diacylglyceryl ethers with a smaller proportion of ether lipids in the phospholipid fraction. In addition, 4-13% of the ingested hexadecanol appeared in the plasma as a material having the chromatographic properties of wax ester. Fatty alcohols were not detected in the plasma as alk-1-enyl lipids.     

Evidence for a H+ nitrate symporter in Aspergillus nidulans

Nitrate transport in Aspergillus nidulans was dependent upon a consistent proton motive force (delta p) across the cell membrane which was maintained in a range of 105 (+/- 6.7) to 131 (+/- 3.4) mV over an external pH span of 5.5 to 7.5. The membrane potential (delta psi) measured by uptake of [3H]-tetra-phenylphosphonium bromide and the transmembrane pH difference (delta pH) measured by the distribution of 3H2O and [14C]- salicylic acid were used to compute the delta p present during transport of nitrate. Energy dependent accumulation of nitrate was measured in actively assimilating and tungstate inhibited cells. A delta G for nitrate of 14 kJ mol-1 was computed from the results. Cells induced for nitrate transport maintained internal nitrate levels of 6 to 8 mM based on an internal volume of 2.6 microliters/mg dry wt as determined by a conventional dual label procedure. A fivefold higher level of cellular nitrate was observed in tungstate inhibited cells. Nitrate accumulation was dependent upon a H+ gradient which was dissipated by treatment with 2-butanol, the ionophores valinomycin and gramicidin and the proton conductors carbonyl cyanide m-chlorophenyl hydrazone and N,N'-dicyclo-hexylcarbodiimide. Significant ATP and nitrate efflux occurred in cells treated with the above agents. The results suggest that nitrate is transported by symport with H+ on a carrier which is functionally linked to a H+ ATPase pump.     

Up-regulation of brain N-methyl-D-aspartate receptors following multiple intracerebroventricular injections of [D-Pen2, D-Pen5] enkephalin and [D-Ala2, Glu4]deltorphin II in mice

The effects of chronic administration of [D-Pen2, D-Pen5]enkephalin and [D-Ala2, Glu4]deltorphin II, the selective agonists of the delta 1- and delta 2-opioid receptors, on the binding of [3H]MK-801, a noncompetitive antagonist of the N-methyl-D-aspartate receptor, were determined in several brain regions of the mouse. Male Swiss-Webster mice were injected intracerebroventricularly (i.c.v.) with [D-Pen2, D-Pen5]enkephalin or [D-Ala2, Glu4]deltorphin II (20 micrograms/mouse) twice a day for 4 days. Vehicle injected mice served as controls. Previously we have shown that the above treatment results in the development of tolerance to their analgesic activity. The binding of [3H]MK-801 was determined in brain regions (cortex, midbrain, pons and medulla, hippocampus, striatum, hypothalamus and amygdala). At 5 nM-concentration, the binding of [3H]MK-801 was increased in cerebral cortex, hippocampus, and pons and medulla of [D-Pen2, D-Pen5]enkephalin treated mice. In [D-Ala2, Glu4]deltorphin II treated mice, the binding of [3H]MK-801 was increased in cerebral cortex and hippocampus. The changes in the binding were due to increases in the Bmax value of [3H]MK-801. It is concluded that tolerance to delta 1- and delta 2-opioid receptor agonists is associated with up-regulation of brain N-methyl-D-aspartate receptors, however, some brain areas affected differ with the two treatments. The results are consistent with the recent observation from this laboratory that N-methyl-D-aspartate receptors antagonists block tolerance to the analgesic action of delta 1- and delta 2-opioid receptor agonists.     

Isotope dilution gas chromatographic-mass spectrometric measurement of tricyclic antidepressant drugs. Utility of the 4-carbethoxyhexafluorobutyryl derivatives of secondary amines

Stable isotope dilution gas chromatographic-mass spectrometric (GC-MS) measurement of tricyclic antidepressants (TCA) is a useful alternative to high-performance liquid chromatography (HPLC) methods when interfering substances prevent accurate quantitation by HPLC. For satisfactory GC-MS analysis, secondary amine TCA must be derivatized. Commonly employed trifluoroacetyl and heptafluorobutyryl derivatives are relatively unstable and cause rapid deterioration of capillary GC columns. Therefore we examined 4-carbethoxyhexafluorobutyryl chloride (CHFB-CI) as an alternative derivatizing agent and developed a stable isotope dilution GC-MS method employing ring-labeled [2H4]-desipramine and [2H4]-imipramine internal standards, which permits measurement of desipramine, nortriptyline, imipramine, and amitriptyline in plasma samples containing one or all of these analytes. The GC-MS assay is linear for each analyte from the lower limit of quantitation (25 ng/mL) up to 1500 ng/mL and correlates well with HPLC measurements. The GC-MS analytic coefficient of variation was 9.7 +/- 1.3% for all analytes considered together. Although interferences are observed in the HPLC assay, thioridazine, perphenazine, cyclobenzaprine, and norcyclobenzaprine do not interfere with GC-MS measurements of the TCA examined here. The stability of the CHFB derivative of secondary amine TCA was found to be superior to that of the trifluoroacetyl derivatives of these compounds.     

Synthesis of nitrodiazirinyl derivatives of neurotoxin II from Naja naja oxiana and their interaction with the Torpedo californica nicotinic acetylcholine receptor

Five singly modified nitrodiazirine derivatives of neurotoxin II (NT-II) from Naja naja oxiana were obtained after NT-II reaction with N-hydroxysuccinimide ester of (2-nitro-4-[3-(trifluoromethyl)-3H-diazirin-3yl]phenoxy)acet ic acid followed by chromatographic separation of the products. To localize the label positions, each derivative was first UV-irradiated and then subjected to reduction, carboxymethylation, and trypsinolysis. Tryptic digests were separated by reversed phase-HPLC, the labeled peptides being identified by mass spectrometry. The derivatives containing the photolabel at the position Lys 25, Lys 26, Lys 44, or Lys 46 were [125I]iodinated by the chloramine T procedure. Each iodinated derivative was found to form photoinduced cross-links with the membrane-bound nicotinic acetylcholine receptor (AChR) from Torpedo californica. The pattern of labeling the receptor's alpha, beta, gamma, or delta subunits was dependent on the photolabel position in the NT-II molecule and differed from that obtained earlier with an analogous series of p-azidobenzoyl derivatives of NT-II. The results obtained indicate that (i) different sides of the neurotoxin molecule are involved in the AChR binding, and (ii) fragments of the different AChR subunits are located close together at the neurotoxin-binding sites.     

Opioid peptide receptor studies. 9. Identification of a novel non-mu- non-delta-like opioid peptide binding site in rat brain

Quantitative binding studies resolved two high-affinity [3H][D-Ala2,D-Leu5]enkephalin binding sites in rat brain membranes depleted of mu binding sites by pretreatment with the irreversible agent BIT. The two binding sites had lower (delta ncx-2, Ki = 96.6 nM) and higher (delta ncx-1, Ki = 1.55 nM) affinity for DPDPE. The ligand-selectivity profile of the delta ncx-1 site was that of a classic delta binding site. The ligand-selectivity profile of the delta ncx-2 site was neither mu- or delta-like. The Ki values of selected agents for the delta ncx-2 site were: [pCl]DPDPE (3.9 nM), DPLPE (140 nM), and DAMGO (2.6 nM). Under these assay conditions, [3H][D-Ala2,D-Leu5]enkephalin binding to the cells expressing the cloned mu receptor is very low and pretreatment of cell membranes with BIT almost completely inhibits [3H]DAMGO and [3H][D-Ala2,D-Leu5]enkephalin binding. Intracerebroventricular administration of antisense DNA to the cloned delta receptor selectively decreased [3H][D-Ala2,D-Leu5]enkephalin binding to the delta ncx-1 site. Administration of buprenorphine to rats 24 h prior to preparation of membranes differentially affected mu, delta ncx-1, and delta ncx-2 binding sites. Viewed collectively, these studies have identified a novel non-mu- non-delta-like binding site in rat brain.     

Postnatal development of delta-opioid receptor subtypes in mice

1. The density and affinity of binding sites for the delta-selective opioid ligands [3H]-[D-Ala2, Asp4]deltorphin (DELT-I), [3H]-[D-Ala2Glu4]-deltorphin (DELT-II), [3H]-[D-Pen2,D-Pen5]enkephalin (DPDPE), and [3H]-naltrindole (NTI) were determined in whole brain from 10, 15, 25 and 60 day-old C57BL mice. 2. At all ages, the analyses of the homologous displacement curves, gave best fits to single rather than to multiple site models. The binding capacity (Bmax) labelled by [3H]-NTI was about one half that labelled by [3H]-DELT-I, [3H]-DELT-II and [3H]-DPDPE. In 25 and 60 day-old mouse brain the DPDPE Bmax was 25% less than the deltorphin-II Bmax. 3. In saturation experiments, specific binding of [3H]-DELT-I on adult mouse brain homogenates was best fitted by a two-site model (34%, high affinity site, Kd = 1.08 nM and 66% low affinity sites, Kd = 39.9 nM). 4. DPDPE produced a biphasic inhibition of specific [3H]-DELTI-I binding, from 15 days of age onwards. The relative percentage of high and low affinity sites was 72% and 28% in 15 day-, 65% and 35% in 25 day- and 30% and 70% in 60 day-old mice. 5. In adult mouse brain labelled with [3H]-DELT-I, DELT-II recognized 71% of high-affinity and 29% of low-affinity sites DELT-I and DPDPE produced monophasic inhibition of specific [3H]-DELT-II binding to brain homogenates of adult mice. 6. These data suggest that a sub-population of delta-sites (probably the delta 2-subtype), recognized by DELT-I, with high affinity for DELT-II and low affinity for DPDPE develops from 25 days onward. 7. In electrically stimulated mouse vas deferens (MVD) the rank order of potency of the three delta-agonists was: DELT-I > DELT-II > DPDPE in 10 day-old mice: and DELT-I- DELT-II > DPDPE, from 25 days onward. During this time, the potency of DELT-II increased about 15 fold whereas the potency of DELT-I and DPDPE increased only 5 times. The higher efficacy of DELT-II could depend on receptor maturation towards the delta 2-subtype.     

Kappa1- and kappa2-opioid receptors mediating presynaptic inhibition of dopamine and acetylcholine release in rat neostriatum

1. The effects of selective opioid receptor agonists and antagonists on N-methyl-D-aspartate (NMDA, 10 microM)-induced release of [3H]-dopamine and [14C]-acetylcholine (ACh) from superfused neostriatal slices were studied to investigate the possible occurrence of functional kappa-opioid receptor subtypes in rat brain. 2. The kappa receptor agonists (-)-ethylketocyclazocine ((-)-EKC), U69593 and the endogenous opioid peptide dynorphin A1-13 caused a naloxone-reversible inhibition of NMDA-induced [3H]-dopamine release, with pD2 values of about 9, 8.5 and 8.2, respectively, whereas both the mu agonist Tyr-D-Ala-Gly-(NMe)Phe-Gly-ol (DAMGO) and the delta agonist D-Pen2-D-Pen5-enkephalin (DPDPE) were ineffective in this respect. The inhibitory effect of submaximally effective concentrations of dynorphin A1-13, U69593 and (-)-EKC on NMDA-induced [3H]-dopamine release were not changed by the delta1/delta2-opioid receptor antagonist naltrindole (up to a concentration of 1 microM, but reversed by the kappa receptor antagonist nor-binaltorphimine (nor-BNI), with an IC50) as low as 0.02 nM, indicating the involvement of U69593-sensitive kappa1-opioid receptors. 3. NMDA-induced [14C]-ACh release was reduced in a naloxone-reversible manner by DPDPE (pD2 about 7.2), dynorphin A1-13 (pD2 6.7) and EKC (pD2 6.2), but not by U69593 and DAMGO. The inhibitory effect of a submaximally effective concentration of DPDPE, unlike those of dynorphin A1-13 and (-)-EKC, on NMDA-induced [14C]-ACh release was antagonized by naltrindole with an IC50 of 1 nM, indicating the involvement of delta-opioid receptors in the inhibitory effect of DPDPE. On the other hand, the inhibitory effects of dynorphin A1-13 and (-)-EKC on [14C]-ACh release were readily antagonized by nor-BNI with an IC50 of about 3 nM. A 100 fold higher concentration of nor-BNI also antagonized the inhibitory effect of DPDPE, indicating the involvement of U69593-insensitive kappa2-opioid receptors in the inhibitory effects of dynorphin A1-13 and (-)-EKC. 4. Although naloxone benzoylhydrazone (NalBzoH), displaying high affinity towards the putative kappa3-opioid receptor, antagonized the inhibitory effects of dynorphin A1-13 and (-)-EKC on [3H]-dopamine and [14C]-ACh release as well as that of U69593 on [3H]-dopamine release, it displayed a low apparent affinity (IC50 about 100 nM) in each case. 5. In conclusion, whereas activation of kappa1-opioid receptors causes presynaptic inhibition of NMDA-induced dopamine release, kappa2 receptor activation results in inhibition of ACh release in rat neostriatum. As such, this study is the first to provide unequivocal in vitro evidence for the existence of functionally distinct kappa-opioid receptor subtypes in the brain.     

Effect of peripherally and centrally administered calcitonin on gallbladder emptying in dogs

The vast molecular heterogeneity of brain gamma-aminobutyric acid type A (GABAA) receptors forms the basis for receptor subtyping. Using autoradiographic techniques, we established the characteristics of cerebellar granule cell GABAA receptors by comparing wild-type mice with those with a targeted disruption of the alpha6 subunit gene. Cerebellar granule cells of alpha6(-/-) animals have severe deficits in high affinity [3H]muscimol and [3H]SR 95531 binding to GABA sites, in agonist-insensitive [3H]Ro 15-4513 binding to benzodiazepine sites, and in furosemide-induced increases in tert-[35S]butylbicyclophosphorothionate binding to picrotoxin-sensitive convulsant sites. These observations agree with the known specific properties of these sites on recombinant alpha6beta2/3gamma2 receptors. In the presence of GABA concentrations that fail to activate alpha1 subunit-containing receptors, methyl-6,7-dimethoxy-4-ethyl-beta-carboline (30 microM), allopregnanolone (100 nM), and Zn2+ (10 microM) are less efficacious in altering tert-[35S]butylbicyclophosphorothionate binding in the granule cell layer of the alpha6(-/-) than alpha6(+/+) animals. These data concur with the deficiency of the cerebellar alpha6 and delta subunit-containing receptors in the alpha6(-/-) animals and could also account for the decreased affinity of [3H]muscimol binding to alpha6(-/-) cerebellar membranes. Predicted additional alterations in the cerebellar receptors of the mutant mice may explain a surplus of methyl-6,7-dimethoxy-4-ethyl-beta-carboline-insensitive receptors in the alpha6(-/-) granule cell layer and an increased diazepam-sensitivity in the molecular layer. These changes may be adaptive consequences of altered GABAA receptor subunit expression patterns in response to the loss of two subunits (alpha and delta) from granule cells.     

Cis-Dichloro(diaminosuccinate diethyl ester)palladium (II) as Pd(II)/Pt(II) model compound for DNA-binding and antitumor properties: solution equilibria of their aqua-, hydroxo-, and/or chloro-species

Cis-Dichloro(diaminosuccinic acid)palladium(II), cis-[Pd(H2dasa)Cl2] (I), or cis-dichloro(diaminosuccinate diethyl ester)palladium(II), cis-[Pd(Et2dasa)Cl2] (II) reacts with two equivalents of AgClO4 to give insoluble Pd(dasa) or an aqueous solution of [Pd(Et2dasa) (H2O)2](ClO4)2, respectively. Three solutions of this salt were titrated with NaOH (I = NaClO4 (0.15M),37 degrees C), and 133 E(H+) data (3.5 < or = pH < or = 7) were treated by SUPERQUAD to fit log beta pqr of cis-aquahydroxo-(pqr = 10-1, -5.25(3)) and di-mu-hydroxo-species (20-2, -6.55(1)). At pH > 7 the ester hydrolysis prevents the calculation of log beta 10-2 for the cis-dihydroxo-complex. Another three solutions of such salt were titrated (I = 0.15M (NaClO4), 37 degrees C) with NaOH and NaCl simultaneously using two potentiometric systems (which measure H+ or Cl-). From 147 E(H+) and E(Cl-) data pairs and the above fixed log beta pqr, SUPERQUAD calculations yield log beta pqr for cis-chloro-aqua (pqr = 110, 3.65(1)), cis-chloro-hydroxo (11-1, -2.68 (4)), and cis-dichloro-species (120, 5.86(3)). Simulated and experimental titrations are in good agreement. Circular dichroism spectra of native DNA and drug:DNA complexes suggest that cis-Pd(H2dasa) and cis-Pd(Et2dasa) chelate moieties induce an opening and rotation of the stacked bases in the double helix. This finding is explained by the abundance of each one and of the total neutral and charged species of II in the tested CD solution.     

Induction of adrenal scavenger receptor BI and increased high density lipoprotein-cholesteryl ether uptake by in vivo inhibition of hepatic lipase

Hepatic lipase (HL) and scavenger receptor type B class I (SR-BI) have both been implicated in high density lipoprotein (HDL)-cholesteryl ester uptake in cholesterol-utilizing tissues. Inactivation of HL by gene-directed targeting in mice results in up-regulation of SR-BI expression in adrenal gland (Wang, N., Weng, W., Breslow, J. L., and Tall, A. R. (1996) J. Biol. Chem. 271, 21001-21004). The net effect on HDL-cholesteryl ester uptake is not known. We determined the impact of acute in vivo inhibition of rat adrenal HL activity by antibodies on SR-BI expression and on human and rat HDL-[3H]cholesteryl ether (CEth) uptake in the adrenal gland. Rat HDL was isolated from rats in which HL activity had been inhibited for 1 h. The rats were studied under basal conditions (not ACTH-treated) and after previous treatment with ACTH for 6 days (ACTH-treated). Intravenous injection of anti-HL resulted in 70% lowering of adrenal HL activity in both conditions which were maintained for at least 8 h. In not ACTH-treated rats, inhibition of adrenal HL increased adrenal SR-BI mRNA (5.2-fold) and mass (1. 6-fold) within 4 h. HL inhibition resulted in 41% and 14% more adrenal accumulation of human HDL-[3H]CEth during 4 and 24 h, respectively. The adrenal uptake of rat HDL-[3H]CEth increased by 68%, 4 h after the antibody injection. ACTH treatment increased total adrenal HL activity from 3.7 +/- 0.5 milliunits to 34.0 +/- 17. 2 milliunits, as well as adrenal SR-BI mRNA from 2.9 +/- 0.7 arbitrary units (A.U.) to 86.8 +/- 41.1 A.U. and SR-BI mass from 7.7 +/- 1.8 A.U. to 63.16 +/- 46.7 A.U. The human HDL-[3H]CEth uptake by adrenals was also significantly increased from 0.58 +/- 0.11% of injected dose to 7.24 +/- 1.58% of injected dose. Inhibition of adrenal HL activity did not result in further induction of SR-BI expression and did not affect human HDL-[3H]CEth uptake. These findings indicate that SR-BI expression may be influenced by changes in HL activity. HL activity is not needed for the SR-BI-mediated HDL-cholesteryl ester uptake by rat adrenal glands.     

Simultaneous determination of diphenhydramine, its N-oxide metabolite and their deuterium-labeled analogues in ovine plasma and urine using liquid chromatography/electrospray tandem mass spectrometry

Our studies on drug disposition in chronically instrumented pregnant sheep involve simultaneous administration of the antihistamine diphenhydramine (DPHM), its deuterated analogue ([2H10]DPHM) and their metabolites to the mother or the fetus via various routes. Such studies require sensitive and selective mass spectrometric methods for quantitation of these labeled and unlabeled compounds in order to assess comparative maternal and fetal drug metabolism. The objective of this study was to develop and validate a liquid chromatographic/tandem mass spectrometric (LC/MS/MS) method for the simultaneous quantitation of DPHM, its N-oxide metabolite and their deuterium-labeled analogues in ovine plasma and urine. Samples spiked with the analytes and the internal standard, orphenadrine, were processed using liquid-liquid extraction. The extract was chromatographed on a propylamino LC column and MS/MS detection was performed in the positive ion electrospray mode using multiple reaction monitoring. The linear concentration ranges of the calibration curves for the N-oxides and the parent amines were 0.4-100.0 and 0.2-250.0 ng ml-1, respectively. In validation tests, the assay exhibited acceptable variability (< or = 15% at analyte concentrations below 2.0 ng ml-1 and < 10% at all other concentrations) and bias (< 15% at all concentrations), and the analytes were stable under a variety of sample handling conditions. Using this method, the labeled and unlabeled N-oxide metabolite was identified in fetal plasma after DPHM and [2H10]DPHM administration. This method will be used further to examine the comparative metabolism of diphenhydramine to its N-oxide metabolite in the mother and the fetus.     

Biological labeling of very low density lipoproteins with cholesteryl linoleyl ether and its fate in the intact rat

In vitro labeling of very low density lipoproteins (VLDL) with radioactive cholesteryl linoleyl ether, an analog of cholesteryl linoleate, was studied. The protocol which gave the highest efficiency and seemed least injurious to the final product included: (1) sonication of the labeled cholesteryl ether with partially delipidated high density lipoproteins (HDL); (2) transfer of the labeled lipids to VLDL in the presence of lipoprotein-deficient human serum; (3) reisolation of the VLDL by ultracentrifugation. Under optimal conditions 70% of the added labeled lipid was recovered with HDL and 60% were transferred from HDL to VLDL. The labeled cholesteryl linoleyl ether was shown to comigrate with the protein of VLDL on agarose gel electrophoresis. In negatively stained preparations, the labeled VLDl and its unlabeled counterpart had similar appearance. The in vitro labeled VLDL was injected into rats and was cleared from the circulation with a t1/2 comparable to endogenously labeled VLDL. More than 80% of the injected dose was recovered in the liver between 3 and 48 h after injection of VLDL labeled with [3H]cholesteryl linoleyl ether of which 91-97% were in the ether form. On radioautography of fixed frozen sections of liver the bulk of the radioautographic reaction was associated with the cytoplasm of hepatocytes. When the VLDL had been labeled also with [14C]cholesteryl linoleate only 35% of injected dose was present in the liver at 3 h, of which 87% was in unesterified form. The distribution of the labeled cholesteryl linoleyl ether, 3-48 h after injection, expressed as per cent of injected dose per organ was 0.7-1.5 in spleen, 0.2-0.5 in lung, 0.1 in heart and 0.2-0.4 in adrenal. The main advantage of the presently described approach in which a nondegradable analog of cholesteryl ester was introduced into VLDL by a biological procedure is the possibility to study the role of various organs to take up circulating cholesteryl ester, especially in species in which LDL is produced from VLDL.     

Tricarboxylic acid cycle in rat brain synaptosomes. Fluxes and interactions with aspartate aminotransferase and malate/aspartate shuttle

The flux through different segments of the tricarboxylic acid cycle was measured in rat brain synaptosomes with gas chromatography-mass spectrometry using either deuterated glutamine or [13C]aspartate. The flux between 2-oxoglutarate and oxaloacetate was estimated to be 3.14 and 4.97 nmol/min/mg protein with and without glucose, respectively. These values were 3-5-fold faster than the flux between oxaloacetate and 2-oxoglutarate (0.92 nmol/min per mg protein) measured in the presence of glucose. The pattern of intermediates labeling suggests that the overall rate-controlling reaction involves either citrate synthase or pyruvate dehydrogenase but not 2-oxoglutarate or isocitrate dehydrogenase. The enrichment in [3,3,4,4-2H4]glutamate from [2,3,3,4,4-2H5]glutamine was as rapid as in [2,3,3,4,4-2H5]glutamate, which indicates that the aspartate aminotransferase reaction is severalfold faster than the flux through the tricarboxylic acid cycle. [13C]Aspartate was rapidly converted to [13C]malate, suggesting that in intact synaptosomes aspartate entry into the mitochondrion is very slow. The finding that aspartate is taken up by mitochondria as malate, along with the observed high enrichment in [3-2H]malate (from [2,3,3,4,4-2H5]glutamine), is consistent with the substantial synaptosomal activity of the malate/aspartate shuttle.     

Herpes simplex virus processivity factor UL42 imparts increased DNA-binding specificity to the viral DNA polymerase and decreased dissociation from primer-template without reducing the elongation rate

A liquid chromatographic/mass spectrometric method to quantitate atorvastatin (AT) and its active metabolites ortho-hydroxy (o-AT) and para-hydroxy (p-AT) atorvastatin in human, dog, and rat plasma was validated. The method consisted of washing plasma samples at high pH with diethyl ether and subsequently extracting the analytes and two internal standards, [d5]-atorvastatin ([d5]-AT) and [d5]-ortho-hydroxy atorvastatin ([d5]-o-AT), from acidified plasma by using diethyl ether. The ether layer was evaporated to dryness and the residue reconstituted in ammonium acetate (20 mM, pH 4.0)-acetonitrile-isopropanol (60:40:1, v/v/v). Chromatographic separation of analytes was achieved by using a YMC J'Sphere H80 (C-18) 150 x 2 mm, 4 microns particle size, column with a mobile phase consisting of acetonitrile-0.1% acetic acid, (70:30, v/v). Analytes were detected by using MS/MS. Sample introduction and ionization was by electrospray ionization in the positive ion mode. The method proved suitable for routine quantitation of AT, o-AT, and p-AT over the concentration range of 0.250 to 25.0 ng/mL. Approximate retention time ranges of p-AT, o-AT, [d5]-o-AT, AT, and [d5]-AT were 2.27 +/- 0.21, 3.36 +/- 0.23, 3.54 +/- 0.46, 4.12 +/- 0.61, and 4.65 +/- 0.65 min, respectively. No peaks interfering with quantitation were observed throughout the validation processes. Mean recoveries of AT, o-AT, and p-AT from plasma ranged 100%-107%, 70.6%-104%, and 47.6%-85.6%, respectively. Mean recoveries of the [d5]-AT and [d5]-o-AT internal standards ranged 98.0%-99.9% and 97.3%, respectively. Interassay precision, based on the percent relative deviation for replicate quality controls for AT, o-AT, and p-AT, was < or = 7.19%, 8.28%, and 12.7%, respectively. Interassay accuracy for AT, o-AT, and p-AT was +/- 10.6%, 5.86%, and 15.8%, respectively. AT, o-AT, and p-AT in human, dog, and rat plasma quality controls were stable to three freeze-thaw cycles. AT, o-AT, and p-AT were stable frozen for 127, 30 and 270 days in human, dog, and rat plasma quality control samples, respectively. Human plasma quality control samples containing AT, o-AT, and p-AT were stable for at least 4 days at ambient room temperature and 37 degrees C. The lower limit of quantitation for all analytes was 0.250 ng/mL for a 1.0-mL sample aliquot.