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Biomedical subjects

D Nikolic

Publications and source records attributed to D Nikolic.

9 recordsLinked to original sources
PubMed1998

Development of a method for quantitation of retinol and retinyl palmitate in human serum using high-performance liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry.

A method for the quantitative analysis of the vitamin A compounds all-trans-retinol and all-trans-retinyl palmitate was developed using high-performance liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (APCI-LC-MS). Unlike previous quantitative mass spectrometric methods for vitamin A, HPLC separations were carried out using a C30 reversed-phase column instead of GC separation. Because no sample hydrolysis or derivatization was necessary, retinyl palmitate was preserved for analysis instead of being hydrolyzed to retinol. Human serum was analyzed following simple hexane extraction without saponification or any additional purification. A comparison of APCI and electrospray ionization showed that only APCI produced a linear response over all four orders of magnitude of retinol and three orders of magnitude of retinyl palmitate concentrations. Selected ion monitoring of the fragment ion of m/z 269 was used for APCI quantitation of both retinol and retinyl palmitate, since it was the base peak and the only abundant ion in the mass spectra of both compounds and the internal standard, retinyl acetate. The ion of m/z 269 corresponded to loss of water, loss of palmitic acid, or elimination of acetic acid from the protonated molecules of retinol, retinyl palmitate and retinyl acetate, respectively. The limit of detection of APCI-LC-MS for all-trans-retinol and all-trans-retinyl palmitate was determined to be approximately 34 fmol/microliter and 36 fmol/microliter (0.670 pmol all-trans-retinol and 0.720 pmol all-trans-retinyl palmitate injected in 20 microliters on-column), respectively. The limit of quantitation was approximately 500 fmol/microliter and 250 fmol/microliter (10 pmol and 5 pmol injected in 20 microliters on-column) for retinol and retinyl palmitate, respectively.

Chromatography, High Pressure Liquid↗
PubMed1998

Alkylation of 2'-deoxynucleosides and DNA by the Premarin metabolite 4-hydroxyequilenin semiquinone radical.

Premarin (Wyeth-Ayerst) is the estrogen replacement treatment of choice and continues to be one of the most widely dispensed prescriptions in the United States. In addition to endogenous estrogens, Premarin contains unsaturated estrogens including equilenin. We synthesized the catechol metabolite of equilenin, 4-hydroxyequilenin (4-OHEN), and found that the semiquinone radical of 4-OHEN reacted with 2'-deoxynucleosides generating very unusual adducts. 2'-Deoxyguanosine (dG), 2'-deoxyadenosine (dA), or 2'-deoxycytosine (dC) all gave four isomers, but no product was observed for thymidine under similar physiological conditions. The structures of these adducts were determined by electrospray mass spectrometry and NMR experiments including 1H, 13C, DQF-COSY, ROESY, HOHAHA, HMQC, and HMBC. The spectral data show that dG forms a cyclic adduct with the 4-OHEN producing 2-N1,3-N2-deoxyguanosyl-1,3-dihydroxy-5,7,9(10)-estratriene-4,17-d ione. Similarly, reaction with dA produced 1-N6,3-C2-deoxyadenosyl-2,3-dihydroxy-5,7,9(10)-estratriene-4,17-d ione, and incubations with dC resulted in 1-N3,3-N4-deoxycytosyl-2,3-dihydroxy-5,7,9(10)-estratriene-4,17-di one. We found that care needed to be taken during the isolation of the dA adducts in particular, as any exposure to acidic environments caused hydrolysis of the sugar moiety leaving alkylated adenine. In mixtures of the deoxynucleosides treated with 4-OHEN, reaction occurred primarily with dG followed by dC and dA. With DNA significant apurinic sites were produced as 4-OHEN-adenine adducts were detected in the ethanol wash prior to hydrolysis. When the DNA was hydrolyzed to deoxynucleosides and analyzed by electrospray mass spectrometry, only one isomer of 4-OHEN-dG and one isomer of 4-OHEN-dC were observed. Our data suggest that several different types of DNA lesions could be expected from 4-OHEN including apurinic sites and bulky stable adducts, in addition to the published oxidized damage to DNA caused by 4-OHEN. The production of these semiquinone radical-derived DNA adducts could play a role in the carcinogenic effects of Premarin estrogens.

Alkylation↗
PubMed1998

The equine estrogen metabolite 4-hydroxyequilenin causes DNA single-strand breaks and oxidation of DNA bases in vitro.

Premarin (Wyeth-Ayerst) is the estrogen replacement treatment of choice and continues to be one of the most widely dispensed prescriptions in North America. In addition to endogenous estrogens, Premarin contains unsaturated equine estrogens, including equilenin [1,3,5(10),6,8-estrapentaen-3-ol-17-one]. In previous work, we showed that the equilenin metabolite 4-hydroxyequilenin (4-OHEN) can be autoxidized to 4-OHEN-o-quinone which readily entered into a redox couple with the semiquinone radical catalyzed by NAD(P)H, P450 reductase, or quinone reductase, resulting in generation of reactive oxygen species [Shen, L., Pisha, E., Huang, Z., Pezzuto, J. M., Krol, E., Alam, Z., van Breemen, R. B., and Bolton, J. L. (1997) Carcinogenesis 18, 1093-1101]. As oxidative damage to DNA by reactive oxygen species generated by redox active compounds has been proposed to lead to tumor formation, we investigated whether 4-OHEN could cause DNA damage. We treated lambda phage DNA with 4-OHEN and found that extensive single-strand breaks could be obtained with increasing concentrations of 4-OHEN as well as increasing incubation times. If scavengers of reactive oxygen species are included in the incubations, DNA could be completely protected from 4-OHEN-mediated damage. In contrast, NADH and CuCl2 enhanced the ability of 4-OHEN to cause DNA single-strand breaks presumably due to redox cycling between 4-OHEN and the semiquinone radical generating hydrogen peroxide and ultimately copper peroxide complexes. We also confirmed that 4-OHEN could oxidize DNA bases since hydrolysis of 4-OHEN-treated calf thymus DNA and HPLC separation with electrospray MS detection revealed oxidized deoxynucleosides, including 8-oxodeoxyguanosine and 8-oxodeoxyadenosine. Our data suggest that DNA single-strand breaks and oxidation of DNA bases by 4-OHEN could contribute to the carcinogenic mechanism(s) of equine estrogens.

Animals↗
PubMed1998

Novel pathway for conversion of chlorohydroxyquinol to maleylacetate in Burkholderia cepacia AC1100.

Burkholderia cepacia AC1100 metabolizes 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) via formation of 5-chlorohydroxyquinol (5-CHQ), hydroxyquinol (HQ), maleylacetate, and beta-oxoadipate. The step(s) leading to the dechlorination of 5-CHQ to HQ has remained unidentified. We demonstrate that a dechlorinating enzyme, TftG, catalyzes the conversion of 5-CHQ to hydroxybenzoquinone, which is then reduced to HQ by a hydroxybenzoquinone reductase (HBQ reductase). HQ is subsequently converted to maleylacetate by hydroxyquinol 1,2-dioxygenase (HQDO). All three enzymes were purified. We demonstrate specific product formation by colorimetric assay and mass spectrometry when 5-CHQ is treated successively with the three enzymes: TftG, TftG plus HBQ reductase, and TftG plus HBQ reductase plus HQDO. This study delineates the complete enzymatic pathway for the degradation of 5-CHQ to maleylacetate.

Bacterial Proteins↗
PubMed1998

Metabolic screening using on-line ultrafiltration mass spectrometry.

An on-line mass spectrometric method has been developed to generate and identify drug metabolites formed by hepatic cytochromes P450. This method, pulsed ultrafiltration-mass spectrometry, may be used for rapid screening of drugs to determine their extent of metabolism by microsomal cytochromes P450 and to characterize the primary metabolites. Rat liver microsomes were trapped in a stirred ultrafiltration chamber fitted with a 100,000 molecular weight cut-off ultrafiltration membrane. A continuous-flow of ammonium acetate buffer was pumped through the chamber and into an electrospray mass spectrometer. Substrates for cytochromes P450 including imipramine, chlorpromazine, and pentoxyresorufin were flow injected through the chamber along with the cofactor, NADPH, and metabolites were detected on-line by using electrospray mass spectrometry. Identical control experiments carried out using boiled microsomes or without NADPH showed no metabolite formation. Naringenin and quinidine, which are inhibitors of some isozymes of cytochrome P450 and are not known to be extensively metabolized, showed no major metabolites. For comparison, imipramine metabolites were also generated by standard batch incubation with microsomes and NADPH, followed by extraction and LC-MS analysis. Similar metabolites were obtained using the flow-through ultrafiltration method and the standard batch microsomal incubation. Tandem mass spectrometry was used to confirm structures of imipramine metabolites including 10-hydroxyimipramine, 2-hydroxyimipramine, imipramine N-oxide, and N-desmethylimipramine. Finally, the feasibility of using ultrafiltration mass spectrometry for high throughput metabolic screening was demonstrated by using on-line mass spectrometry for only 3 min per incubation instead of monitoring the entire elution profile. By carrying out multiple ultrafiltration experiments in parallel, efficient use of the mass spectrometric detector may be obtained with a throughput of at least 20 incubations per hour. Throughputs of up to 60 profiles per hour should be possible. On-line ultrafiltration electrospray mass spectrometry offers a streamlined, higher-throughput method for in vitro formation and mass spectrometric characterization of microsomal drug metabolites.

Animals↗
PubMed1997

Screening solution-phase combinatorial libraries using pulsed ultrafiltration/electrospray mass spectrometry.

A method is described whereby a family of homologues is synthesized in a one-pot reaction, without isolation or purification, and the reaction mixture is screened using a competitive binding assay based on pulsed ultrafiltration/electrospray mass spectrometry (PUF/ESMS) to tentatively identify those derivatives having the highest affinity for a target receptor. As a model system to test this approach, a synthetic scheme designed to prepare a series of analogues of the adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), as diastereomeric mixtures, was carried out. Pulsed ultrafiltration screening of the crude reaction mixture against controls without protein detected protonated molecules corresponding to EHNA-type derivatives and three of its linear, alkyl homologues but did not show protonated molecules for an isobutyl or benzylic EHNA derivative, suggesting the latter was inactive. To verify this conclusion, we prepared E/THNA, the linear homologues, and the benzylic derivative (each as a diastereomeric mixture) and bioassayed them for them adenosine deaminase inhibition index ([I]/[S]0.5). The bioassay results for the individually synthesized analogues were in good agreement with that predicted by the observed relative ion enhancement in the PUF experiments. Thus, the PUF protocol might be used as a general method to quickly provide direction to the chemist in search of drug candidates.

Adenine↗
PubMed1997

Pulsed ultrafiltration mass spectrometry: a new method for screening combinatorial libraries.

In response to the need for rapid screening of combinatorial libraries to identify new lead compounds during drug discovery, we have developed an on-line combination of ultrafiltration and electrospray mass spectrometry, called pulsed ultrafiltration mass spectrometry, which facilitates the identification of solution-phase ligands in library mixtures that bind to solution-phase receptors. After ligands contained in a library mixture were bound to a macromolecular receptor, e.g., human serum albumin or calf intestine adenosine deaminase, the ligand-receptor complexes were purified by ultrafiltration and then dissociated using methanol to elute the ligands into the electrospray mass spectrometer for detection. Ligands with dissociation constants in the micromolar to nanomolar range were successfully bound, released, and detected using this method, including warfarin, salicylate, furosemide, and thyroxine binding to human serum albumin, and erythro-9-(2-hydroxy-3-nonyl)adenine binding to calf intestine adenosine deaminase. Repetitive bind- and-release experiments demonstrated that the receptor could be reused. Thus, pulsed ultrafiltration mass spectrometry was shown to provide a simple and powerful new method for the screening of combinatorial libraries in support of new drug discovery.

Adenine↗