Deuterium-labeled Analogs Research Articles

Surprising Structural Lability of a Cysteine‐S‐Conjugate Precursor of 4‐Methyl‐4‐sulfanylpentan‐2‐one, a Varietal Aroma in Wine of Vitis vinifera L. cv. Sauvignon Blanc

4-Methyl-4-sulfanylpentan-2-one (1; 4MSP) provides a characteristic aroma compound of wines made from Vitis vinifera L. cv. Sauvignon blanc. 4MSP has a strong box-tree odor with a very low perception threshold and is derived from the cysteinylated precursor S-(1,1-dimethyl-3-oxobutyl)cysteine (4; P-4MSP). P-4MSP is transformed into 4MSP during alcoholic fermentation and is an excellent marker of varietal aroma potential. An improved synthesis of P-4MSP as well as of its deuterium-labeled analogue [D(6)]-P-4MSP is described. Several analytical methods (NMR, IR, LSI-MS, GC/MS, ESI-MS(n)) were combined to elucidate spontaneous reversible structural changes of P-4MSP at different pH values. At low pH, P-4MSP has a linear keto form. The keto-enol tautomerism was observed at neutral pH. At pH 8, the formation of N-substituted intramolecular hemiaminal was characterized by ESI-MS and ESI-MS(n) experiments. The hemiaminal loses H(2)O at high pH to produce a cycloimine, which is easily opened by acid hydrolysis. The keto-enol tautomerism explained the incorporation of only six D-atoms during the preparation of the P-4MSP deuterated standard even if [D(10)]mesityl oxide was used. Derivatization conditions for GC/MS analysis strongly affected the ratio of the monosilylated intramolecular cyclic form and the disilylated linear form of P-4MSP. The structural changes of P-4MSP may have a considerable impact on the development of methods of measuring varietal aroma potential.

Electrospray LC-MS Method with Solid-Phase Extraction for Accurate Determination of Morphine-, Codeine-, and Ethylmorphine-Glucuronides and 6-Acetylmorphine in Urine

A method for the identification and quantification of morphine-3-glucuronide, codeine-6-glucuronide, ethylmorphine-6-glucuronide, and 6-acetylmorphine in human urine based on solid-phase extraction (SPE) and electrospray ionization liquid chromatography-mass spectrometry (LC-MS) was validated for use as a confirmation procedure in combination with immunochemical screening for opiates. Three deuterium-labelled analogues were used as internal standards: morphine-3-glucuronide-d3, codeine-d3, and 6-acetylmorphine-d3. Fifty-microliter aliquots of urine were prepared by SPE using 30-mg Oasis HLB cartridges. The chromatographic system consisted of a 2.0 x 100-mm C18 column and the gradient elution buffers used acetonitrile and 25 mmol/L formic acid. The protonated molecular ions were monitored in the selected ion monitoring mode together with one qualifier ion for each analyte. The interassay variability was less than 10% at the reporting limit 30 ng/mL for 6-acetylmorphine and 300 ng/mL for the other analytes. The method was validated by comparison with a reference gas chromatographic (GC)-MS method using authentic urine samples. The two methods agreed completely regarding identified analytes, and for the quantitative results there were slightly lower levels when measuring glucuronides directly as compared to total determination after hydrolysis by GC-MS. This result was to be expected because the free compounds are not measured with the LC-MS method. This study concludes that the presented LC-MS method is robust and reliable, and suitable for use as a confirmation method in clinical urine drug testing for opiates.

Rapid measurement of deuterium-labeled long-chain fatty acids in plasma by HPLC-ESI-MS

Imbalanced fatty acid metabolism contributes significantly to the increased incidence of metabolic disorders. Isotope-labeled fatty acids (2H, 13C) provide efficient means to trace fatty acid metabolism in vivo. This study reports a new and rapid method for the quantification of deuterium-labeled fatty acids in plasma by HPLC-MS. The sample preparation protocol developed required only hydrolysis, neutralization, and quenching steps followed by high-performance liquid chromatography-electrospray ionization-mass spectrometry analysis in negative ion mode using single ion monitoring. Deuterium-labeled stearic acid (d7-C18:0) was synthesized to reduce matrix interference observed with d5 analog, which improved the limit of detection (LOD) significantly, depending on the products analyzed. Linearity > 0.999 between the LOD (100 nM) and 30 microM, accuracy > 90%, precision > 88%, and adequate recovery in the dynamic range were obtained for d7-C18:0 and d7-oleic acid (C18:1). Upon oral dosing of d7-C18:0 in rats, the parent compound and its desaturation and beta-oxidation products, d7-C18:1 and d7-C16:0, were circulating with a maximal concentration ranging from 0.6 to 2.2 microM, with significant levels of d7-fatty acids detected for up to 72 h.

Metabolism and Bioactivation of 3-Methylindole by Human Liver Microsomes

Metabolism and bioactivation of 3-methylindole (3MI) were investigated in human liver microsomes. The metabolism of two deuterium-labeled analogues of 3MI permitted a relatively unambiguous identification of multiple metabolites and glutathione (GSH) adducts of reactive intermediates. A total of eight oxidized metabolites were detected, five of which were assigned as previously identified 3-methyloxindole, 3-hydroxy-3-methylindolenine, 3-hydroxy-3-methyloxindole, 5-hydroxy-3-methylindole, and 6-hydroxy-3-methylindole. Among the three new metabolites, one was either 4- or 7-OH-3-methylindole, and the other two were derived from additional oxidation on the phenyl ring of 3-methyloxindole. When GSH was added to the microsomal incubations, seven conjugates that had molecular ions corresponding to the incorporation of GSH and an atom of oxygen at m/z 453 (group I) were produced, and two additional conjugates had molecular ions at m/z 437 that corresponded to the incorporation of GSH with no additional oxygen (group II). Two conjugates in group I (m/z 453) were apparently derived by GSH addition to the 5,6-epoxide metabolite of 3-methyloxindole. These two GSH adducts were tentatively identified as 5-(glutathione-S-yl)-3-methyloxindole and 6-(glutathione-S-yl)-3-methyloxindole. The most abundant conjugate in group I was identified as 3-(glutathione-S-yl)-3-methyloxindole, which substantiated the presence of the putative 2,3-epoxy-3-methylindole intermediate. The remaining four adducts in group I were likely formed by conjugation of GSH at different positions of the phenyl ring, possibly via oxidation of 5-hydroxy-3-methylindole and 6-hydroxy-3-methylindole to two very interesting new electrophilic benzoquinone imine intermediates. For the group II conjugates (m/z 437), two isomers were identified as 2-(glutathione-S-yl)-3-methylindole and 3-(glutathione-S-yl-methyl)-indole. The former adduct was primarily derived from the 2,3-epoxide intermediate by thiol conjugation followed by dehydration. The latter adduct was consistent with our previously published work on the dehydrogenation of 3MI. In those studies, we showed that the reactive intermediate, 3-methylenenindolenine, was formed by hydrogen abstraction at the methyl group and was trapped with GSH. The putative dehydrogenation bioactivation mechanism is also substantiated by the finding that CYP2E1 selectively generated 2-(glutathione-S-yl)-3-methylindole but did not produce 3-(glutathione-S-yl-methyl)-indole. In summary, the results not only confirmed the formation of 2,3-epoxide-3-methylindole in human liver microsomes but also suggested that the phenolic metabolites of 3-methylindole were dehydrogenated to previously uncharacterized reactive intermediates.

Thermal Reactions of 7-d- and 8-d-Bicyclo[4.2.0]oct-2-enes

The gas phase thermal reactions exhibited by bicyclo[4.2.0]oct-2-ene and 7-d and 8-d analogues at 300 degrees C have been followed kinetically through GC and 2H NMR spectroscopic analyses. In contrast to the pattern of transformations exhibited by bicyclo[3.2.0]hept-2-ene and deuterium-labeled analogues, no reactions initiated by C1-C6 bond cleavage are seen, epimerization at C8 is much faster than [1,3] shifts leading to bicyclo[2.2.2]oct-2-ene, and the ratio of rate constants for [1,3] carbon migration with inversion versus migration with retention is approximately 1.4. Homolysis of C1-C8 to give a conformationally flexible diradical intermediate having a relatively long lifetime and multiple options for further reaction (re-formation of C1-C8 with or without net epimerization, fragmentation to 1,3-cyclohexadiene and ethylene, migration to the original C3 with inversion or retention) accords well with the observations. Clearly, orbital symmetry control does not govern stereochemistry for the [1,3] sigmatropic carbon shifts.

Investigation of low‐abundant in vitro metabolites of stable isotope‐labelled BAL4815 by accurate mass capillary‐LC‐ESI‐qTof‐MS and MS/MS

The metabolic profile of BAL4815, an antifungal azole drug, was determined using in vitro rat hepatocyte incubations and subsequent analysis by capillary LC-qTof-MS and MS/MS including accurate mass determination. For the detection of the metabolites, a mixture of the drug and its deuterium-labelled analogue was used for incubations. Metabolic stability of BAL4815 was high in cultured rat hepatocytes. However, several low-abundant metabolites were detected by the use of capillary LC-qTof-MS and manual investigation of the data. The peak intensity of the most abundant metabolite was close to the limit of detection. Except for an apparent oxidation product, the masses of the other detected metabolites could not be assigned to a single and frequently occurring biotransformation. Accurate mass determination and possible elemental compositions suggested that metabolism occurred through a combination of glutathionylation and defluorination. This was verified using accurate mass MS/MS. The use of accurate mass measurements and the derived suggestions for the elemental compositions were essential to elucidate this atypical metabolic pathway. A mass accuracy better than 8 ppm could be achieved for most assigned MS and MS/MS signals with intensities less than 6 cps in the spectra.

Structural Analysis of Ionic Organotin(IV) Compounds Using Electrospray Tandem Mass Spectrometry

A novel electrospray ionization (ESI) mass spectrometric approach for the structure elucidation of ionic organotin(IV) compounds or complexes with weakly bonded ligands as for example monodentate carboxylates or sulfonates is proposed using both positive-ion and negative-ion ESI tandem mass spectra. The ionization mechanism of organotin(IV) compounds involves the cleavage of the most labile bond with an ionic character yielding two complementary ions, [Cat]+ and [An]-. Positively charged species containing tin atom, [Cat]+, are analyzed in the positive-ion mode and negatively charged species without the tin atom, [An]-, in the negative-ion mode. Fragmentation patterns of [C24H29N2Sn]+, [C21H22NSn]+, and [C17H30NSn]+ ions are proposed based on the detailed interpretation of MSn spectra, which is simplified by an easy recognition of characteristic tin isotopic clusters in particular fragment ions. Proposed fragmentation mechanisms are supported by comparison with MSn spectra of deuterium-labeled analogues. The applicability of this method is illustrated on two sets of organotin(IV) compounds, including seven [2,6-bis(dimethylaminomethyl)phenyl]diphenyltin(IV) derivatives with small inorganic counteranions X (Br, NO3, SCN, BF4, SeCN, CN, PF6), six organotin(IV) complexes containing two C,N-chelating ligands with azo dyes, and the identification of unknown hydrolysis products.

Tandem mass spectrometric analysis of cyclophosphamide, ifosfamide and their metabolites

A detailed multi-stage (MSn) fragmentation study of cyclophosphamide (CP), ifosfamide (IF) and their major metabolites, using an ion-trap mass spectrometer and a Q-TOF mass spectrometer, was performed with the aid of specifically deuterium-labeled analogs. The analytes showed good responses in positive-ion electrospray mass spectrometry as [MH]+ ions. Tandem mass spectra revealed a wealth of structurally specific ions, allowing characterization of the fragmentation pathways of these analytes. The major fragmentation pathways of the protonated CP and IF are elimination of ethylene from C5 and C6 of 1,3,2-oxazaphosphorine-2-oxide via a McLafferty rearrangement, and cleavage of the P-N bond. However, their activated 4-OOH and 4-OH metabolites primarily underwent hydrogen peroxide elimination and dehydration, respectively, followed by fragmentation pathways similar to those of CP and IF. These results should prove useful in structural elucidation of future analogs of CP and IF, and/or of their metabolites.

Screening for unknown synthetic steroids in human urine by liquid chromatography‐tandem mass spectrometry

Chemically modified steroids (designer steroids), including tetrahydrogestrinone and norbolethone, pose a threat to the integrity of the sport community. These compounds have recently been detected in urine specimens from athletes, resulting in temporary or permanent suspension from amateur and/or professional competition. Triple quadrupole mass spectrometers enable doping control laboratories to screen for unknown, anabolic, androgenic steroids utilizing precursor ion scans. On the basis of common dissociation patterns of steroids with common structural features, characteristic product ions were selected to serve as diagnostic markers for previously unidentified drugs or drug metabolites in human urine samples. An assay was established to complement standard screening procedures. Urine specimens were enzymically hydrolyzed, partitioned into ether, concentrated, and analyzed by precursor ion scanning. Spectra from samples fortified with eight standard compounds (methyltestosterone, ethyltestosterone, 1-testosterone, gestrinone, dihydrogestrinone, tetrahydrogestrinone, norbolethone, and propyltrenbolone) and one deuterium-labeled analog (d(4)-tetrahydrogestrinone) at 50 ng/ml of urine, had precursor ion peaks other than those from common endogenous steroids. Subsequent product ion scan experiments on precursor ions of peaks of unknown origin provided structural identification of the unknown compounds.

Mass spectrometry of 1,2,5-oxadiazole N-oxide derivatives: use of deuterated analogues in fragmentation pattern studies

This paper reported on the study of fragmentation pattern in mass spectrometry of 1,2,5-oxadiazole N-oxide derivatives involving deuterium-labeled analogues to identify some critical fragmentations. A neutral CH2O loss from 3-hydroxymethyl-N2-oxide-4-phenyl-1,2,5-oxadiazole was confirmed with the corresponding mono-deuterated analogue. An OH loss, involving the oxygen of N-oxide, via beta-H and delta-H rearrangement, was clearly revealed from 3-(4-methylpiperazine-1-ylmethyl)-N2 -oxide-4-phenyl-1,2,5-oxadiazole using the adequate tetra-deuterated analogue. N-oxide isomer and deoxygenated analogues were also used to confirm the participation of the oxide moiety in the fragmentation process.

Quantitative liquid chromatography–tandem mass spectrometric analysis of 11-dehydroTXB2 in urine

A simple and selective determination method of 11-dehydrothromboxane B 2 (11-dehydroTXB 2 ), which is urinary metabolite of TXA 2 , has been developed employing liquid chromatography–tandem mass spectrometry (LC–MS–MS). 11-DehydroTXB 2 and its deuterium-labeled analogue as an internal standard were extracted from urine by simple solid-phase extraction (SPE). These compounds were analyzed using LC–MS–MS in the selected reaction monitoring (SRM) mode, by monitoring the transitions from m / z 367 to m / z 161 for 11-dehydroTXB 2 and from m / z 371 to m / z 165 for its internal standard. A good linear response over the range 50 pg–10 ng per tube was demonstrated. The values determined by LC–MS–MS were well validated and closely corresponded to the values determined by gas chromatography–mass spectrometry (GC–MS). The mean concentration of 11-dehydroTXB 2 in urine of healthy adults was 635±427 pg/mg creatinine (mean±S.D., n =13). This simple, accurate and selective determination method described in this study should greatly aid in evaluating the role of TXA 2 in vivo.

Quantitative liquid chromatography–tandem mass spectrometric analysis of 11-dehydroTXB 2 in urine

A simple and selective determination method of 11-dehydrothromboxane B 2 (11-dehydroTXB 2), which is urinary metabolite of TXA 2, has been developed employing liquid chromatography–tandem mass spectrometry (LC–MS–MS). 11-DehydroTXB 2 and its deuterium-labeled analogue as an internal standard were extracted from urine by simple solid-phase extraction (SPE). These compounds were analyzed using LC–MS–MS in the selected reaction monitoring (SRM) mode, by monitoring the transitions from m/ z 367 to m/ z 161 for 11-dehydroTXB 2 and from m/ z 371 to m/ z 165 for its internal standard. A good linear response over the range 50 pg–10 ng per tube was demonstrated. The values determined by LC–MS–MS were well validated and closely corresponded to the values determined by gas chromatography–mass spectrometry (GC–MS). The mean concentration of 11-dehydroTXB 2 in urine of healthy adults was 635±427 pg/mg creatinine (mean±S.D., n=13). This simple, accurate and selective determination method described in this study should greatly aid in evaluating the role of TXA 2 in vivo.

Dormancy termination of western white pine ( Pinus monticola Dougl. Ex D. Don) seeds is associated with changes in abscisic acid metabolism

Western white pine (Pinus monticola) seeds exhibit deep dormancy at maturity and seed populations require several months of moist chilling to reach their uppermost germination capacities. Abscisic acid (ABA) and its metabolites, phaseic acid (PA), dihydrophaseic acid (DPA), 7'-hydroxy ABA (7'OH ABA) and ABA-glucose ester (ABA-GE), were quantified in western white pine seeds during dormancy breakage (moist chilling) and germination using an HPLC-tandem mass spectrometry method with multiple reaction monitoring and internal standards incorporating deuterium-labeled analogs. In the seed coat, ABA and metabolite levels were high in dry seeds, but declined precipitously during the pre-moist-chilling water soak to relatively low levels thereafter. In the embryo and megagametophyte, ABA levels decreased significantly during moist chilling, coincident with an increase in the germination capacity of seeds. ABA catabolism occurred via several routes, depending on the stage and the seed tissue. Moist chilling of seeds led to increases in PA and DPA levels in both the embryo and megagametophyte. Within the embryo, 7'OH ABA and ABA-GE also accumulated during moist chilling; however, 7'OH ABA peaked early in germination. Changes in ABA flux, i.e. shifts in the ratio between biosynthesis and catabolism, occurred at three distinct stages during the transition from dormant seed to seedling. During moist chilling, the relative rate of ABA catabolism exceeded ABA biosynthesis. This trend became even more pronounced during germination, and germination was also accompanied by a decrease in the ABA catabolites DPA and PA, presumably as a result of their further metabolism and/or leaching/transport. The transition from germination to post-germinative growth was accompanied by a shift toward ABA biosynthesis. Dormant imbibed seeds, kept in warm moist conditions for 30 days (after an initial 13 days of soaking), maintained high ABA levels, while the amounts of PA, 7'OH ABA, and DPA decreased or remained at steady-state levels. Thus, in the absence of conditions required to break dormancy there were no net changes in ABA biosynthesis and catabolism.

A method for profiling classes of plant hormones and their metabolites using liquid chromatography-electrospray ionization tandem mass spectrometry: an analysis of hormone regulation of thermodormancy of lettuce (Lactuca sativa L.) seeds.

A highly selective and sensitive method for the simultaneous analysis of several plant hormones and their metabolites is described. The method combines high-performance liquid chromatography (HPLC) with positive and negative electrospray ionization-tandem mass spectrometry (ESI-MS/MS) to quantify a broad range of chemically and structurally diverse compounds. The addition of deuterium-labeled analogs for these compounds prior to sample extraction permits accurate quantification by multiple reaction monitoring (MRM). Endogenous levels of abscisic acid (ABA), abscisic acid glucose ester (ABA-GE), 7'-hydroxy-abscisic acid (7'-OH-ABA), phaseic acid (PA), dihydrophaseic acid (DPA), indole-3-acetic acid (IAA), indole-3-aspartate (IAAsp), zeatin (Z), zeatin riboside (ZR), isopentenyladenine (2iP), isopentenyladenosine (IPA), and gibberellins (GA)1, GA3, GA4, and GA7 were determined simultaneously in a single run. Detection limits ranged from 0.682 fmol for Z to 1.53 pmol for ABA. The method was applied to the analysis of plant hormones and hormonal metabolites associated with seed dormancy and germination in lettuce (Lactuca sativa L. cv. Grand Rapids), using extracts from only 50 to 100 mg DW of seed. Thermodormancy was induced by incubating seeds at 33 degrees C instead of 23 degrees C. Germinating seeds transiently accumulated high levels of ABA-GE. In contrast, thermodormant seeds transiently accumulated high levels of DPA after 7 days at 33 degrees C. GA1 and GA3 were detected during germination, and levels of GA1 increased during early post-germinative growth. After several days of incubation, thermodormant seeds exhibited a striking transient accumulation of IAA, which did not occur in seeds germinating at 23 degrees C. We conclude that hormone metabolism in thermodormant seeds is surprisingly active and is significantly different from that of germinating seeds.

The importance of chromatographic separation in LC/MS/MS quantitation of drugs in biological fluids: detection, characterization, and synthesis of a previously unknown low-level nitrone metabolite of a substance P antagonist.

The observation of an interference peak in plasma samples from dogs dosed with compound I led to the discovery of an unidentified metabolite. The unknown metabolite had the same molecular weight as the parent drug, and their fragmentation profiles were also quite similar. LC/MS/ MS analysis of the plasma extracts of dogs and rats dosed with I and its deuterium-labeled analogue suggested a nitrone structure for the unknown metabolite. Synthesized nitrone matched the unknown metabolite with identical retention time and nearly identical fragmentation profile. The nitrone slowly decomposed in acidic aqueous solution at ambient temperature and also underwent in-source, thermal-induced hydrolysis during electrospray ionization mass spectrometric analysis. The reaction of the nitrone with diethyl acetylenedicarboxylate readily generated a [2 + 3] cycloaddition product. The example shown here clearly demonstrates that precautions must be taken when LC/MS/MS quantitation is conducted in the selected reaction monitoring mode.

Determination of 4-hydroxy-3-methoxyphenylethylene glycol 4-sulfate in human urine using liquid chromatography-tandem mass spectrometry.

A major metabolite of norepinephrine (NE) in brain is 4-hydroxy-3-methoxyphenylethylene glycol (MHPG). In many species, a large fraction of MHPG formed in brain is converted to the sulfate conjugate. Consequently, MHPG sulfate has been proposed as a biomarker for NE metabolism in the central nervous system. As part of the clinical trials of the monoamine oxidase inhibitor selegiline for treating cocaine addiction, we required a method for measuring urine concentrations of MHPG sulfate. Using a deuterium-labeled analogue as an internal standard, we developed a liquid chromatography-electrospray ionization tandem mass spectrometry (LC-MS/ MS) method for determination of MHPG sulfate in human urine. Sample preparation involves simply diluting 50 microL of urine with 1 mL of ammonium formate buffer and adding the internal standard. The sample is centrifuged, the supernate is transferred to an autosampler vial, and 10 microL is injected into the LC-MS/MS system. Standard curves from 50 to 10,000 ng/mL are generated. Only one sample of 277 clinical samples analyzed had a concentration outside of this range. Precision (coefficient of variation) ranged from 1.9 to 9.7%, and accuracy ranged from 97 to 103% of expected values for controls prepared by spiking sulfatase-treated urine with MHPG sulfate.

Electrospray ionization and tandem mass spectrometry of cysteinyl eicosanoids: leukotriene C 4 and FOG 7

The cysteinyl leukotrienes, LTC 4, LTD 4 and LTE 4, and the recently described cysteinyl eicosanoid, 5-oxo-7-glutathionyl-8,11,14-eicosatrienoic acid (FOG 7) have been analyzed by tandem mass spectrometry. Both [M−H] − and [M+H] + ions were produced by electrospray ionization and collision-induced dissociation of these molecular ion species were studied using both an ion trap and a triple quadrupole instrument. Product ion spectra obtained were characteristic of the structure of the cysteinyl leukotrienes and mechanisms of ion formation were investigated by using deuterium-labeled analogs. The product ion spectrum obtained following collision-induced dissociation of the [M−H] − anion from FOG 7 was devoid of significant structural information and further studies of collision activation of the [M+H] + spectrum were therefore examined. Positive ion MS 3 spectra obtained in the ion trap from the γ-glutamate cleavage products of FOG 7 and its derivative (d 7-FOG 7) afforded an abundant ion not observed in spectra generated from the cysteinyl leukotrienes. Formation of this fragment ion likely occurred via a McLafferty-type rearrangement to afford cleavage of the C6–C7 bond adjacent to the sulfur atom and was valuable for the identification of the structure of FOG 7 and defining the biosynthetic pathway as a 1,4-Michael addition of glutathione to 5-oxo-eicosatetraenoic acid (5-oxo-ETE).

Heparin reacts with and inactivates nitric oxide.

Although heparin is a well-known anticoagulant, in some cases it promotes a prothrombotic state and does so through both antibody-dependent and antibody-independent platelet activation. In this study, heparin was found to reverse the antiplatelet effect of an NO donor. S-nitroso-glutathione (SNO-Glu), with an EC(50) of 1.8 U/mL. Ultraviolet/visible spectral analysis and the Griess assay showed that increasing heparin concentrations on a dose-dependent basis eliminated acidified NO(x) species. Since heparin is a heterogeneous mixture of glycosaminoglycans, the effects of six different heparin disaccharides were compared with various substitutions on the hexose rings to determine which functional group(s) of the polysaccharide interact with acidified NO(x). Among the six disaccharides tested, only types I-S and II-S had the effect, suggesting that the sulfamino-group at the C2 position of the glucosamine moiety was critical for the elimination of acidified NO(x) species. Mass spectrometry experiments gave results consistent with these observations, indicating that only the I-S and II-S heparin disaccharides were modified upon treatment with NaNO(2)/HCl. Negative-ion electro-spray ionization MS and tandem MS analyses of the native compounds and their deuterium-labeled analogs confirmed that the reaction products from nitrosation of these N-sulfated disaccharides had eliminated the C2-sulfamino-moiety and replaced it with methoxide derived from the solvent. Participation of the 6-sulfato-substituent appears to facilitate the elimination reaction. These data show that heparin can impair the antiplatelet properties of nitric oxide by interacting with the nitrosating species, and suggest that heparin-like glycosamino-glycans may interact with endothelium-derived nitric oxide in vivo to regulate the bioactivity of this important antiplatelet and vasorelaxant substance.

Metabolism of equilenin in MCF-7 and MDA-MB-231 human breast cancer cells.

Sulfate conjugates of the B-ring unsaturated estrogens, equilin, equilenin, and 8-dehydroestrone, and their 17alpha- and 17beta-dihydro analogues, constitute about 54% of Premarin (Wyeth-Ayerst), the most commonly prescribed estrogen formulation in estrogen replacement therapy. Despite the wide clinical use of Premarin, there have been very few studies on the metabolism of the B-ring unsaturated estrogens in humans and there is no information regarding the fate of these compounds in breast tissue or tumors. In this study, we investigated the metabolism of equilenin in two lines of human breast-cancer cells, MCF-7 and MDA-MB-231. MCF-7 cells respond to treatment with Ah-receptor agonists with induction of cytochromes P450 1A1 and 1B1, whereas in MDA-MB-231 cells P450 1B1 is predominantly induced. Metabolites of equilenin were identified and quantified by GC/MS utilizing a series of synthetic metabolite standards and deuterium-labeled analogues as internal standards. In the two cell lines, the same pathways of equilenin metabolism were observed. Equilenin was reduced at C-17 to the 17beta-dihydro form, with minimal production of the 17alpha-dihydro isomer. Both equilenin and 17beta-dihydroequilenin were hydroxylated at the C-4 position, and the resultant catechol metabolites were methylated to form 4-methoxyequilenin and 4-methoxy-17beta-dihydroequilenin. Rates of equilenin metabolism were markedly elevated in cultures exposed to the Ah-receptor agonists, 2,3,7,8-tetrachlorodibenzo-p-dioxin and 3,4,4',5-tetrachlorobiphenyl, implicating the activities of P450s 1A1 and 1B1 in the metabolism. The 2-hydroxylation pathways of equilenin and 17beta-dihydroequilenin metabolism were not observed. In microsomal reactions with cDNA-expressed human enzymes, both P450s 1A1 and 1B1 catalyzed the 4-hydroxylation of 17beta-dihydroequilenin, whereas with 17beta-estradiol as substrate P450 1A1 catalyzes predominantly 2-hydroxylation and P450 1B1 predominantly 4-hydroxylation. Since P450 1B1 is constitutively expressed and both P450s 1A1 and 1B1 are inducible in many extrahepatic tissues including the mammary epithelium, these results indicate the potential for 4-hydroxylation of equilenin and 17beta-dihydroequilenin in extrahepatic, estrogen-responsive tissues.

Quantification of Neurosteroids in Rat Plasma and Brain Following Swim Stress and Allopregnanolone Administration Using Negative Chemical Ionization Gas Chromatography/Mass Spectrometry

A simplified method for the quantitative analysis of neurosteroids in rat plasma and brain is described. The method uses negative chemical ionization gas chromatography/mass spectrometry and involves the synthesis of pentafluorobenzyloxime/trimethylsilyl ether derivatives with excellent chromatographic and electron-capturing properties. Deuterium-labeled analogs of the steroids of interest were synthesized and used as internal standards. The steroids (allopregnanolone, epiallopregnanolone, pregnenolone, testosterone, and dehydroepiandrosterone) were isolated from the plasma or brain matrix by a rapid and straightforward solid-phase extraction procedure. The mass spectrometer was operated in a selective ion monitoring mode, allowing for picograms of neurosteroids to be quantified from biological extracts. The method was linear (typical R2 = 0.999) over the concentration range (100 to 8000 pg from 0.3 ml plasma and 250 to 8000 pg from 100 mg brain tissue) with good precision and accuracy. In experimental protocols, the procedure was suitable for measuring concentrations of endogenous neurosteroids in rat plasma and brain. Significant elevations (P < 0.001) were observed in the frontal cortex for allopregnanolone and pregnenolone following a swim stress and for allopregnanolone and epiallopregnanolone following allopregnanolone injection (8 mg/kg, sc). The present method allows accurate determination of neurosteroids and will be helpful in elucidating the role of neurosteroids in health and disease.