High-performance liquid chromatography of proteins

Incubation of liver microsomes with GDP [14C] mannose leads to the formation of lipid-linked derivatives of [14C] mannose, a dolichol phosphate monosaccharide and dolichol pyrophosphate oligosaccharides. Standard procedures for separating these two types of compounds from each other were found to be deficient in that fractions thought to contain only dolichol pyrophosphate oligosaccharides are contaminated with dolichol phosphate mannose. This paper presents a column chromatographic procedure which conveniently separates the products of an 8 min labeling experiment into two components; dolichol phosphate [14C]mannose and a [14C]-mannose containing oligosaccharide which is also lipid bound. When this oligosaccharide is released from the lipid by hydrolysis and chromatographed on Sephadex G-50 or G-15 it gives a single peak with an indicated molecular weight of 1100. However, when this released oligosaccharide is chromatographed on concanavalin A Sepharose it is resolved into two peaks suggesting that there may be 2 oligosaccharide of approximately the same size but different structures. After brief periods of labeling with GDP [14C]mannose (5 s) an additional oligosaccharide of 3 to 4 sugar residues can be found in the dolichol pyrophosphate oligosaccharides fraction. Incubation of liver microsomes with UDP [14C]glucose or UDP[14C]galactose produces oligosaccharide components containing 7--8 sugar residues. Labeling of microsomes with UDP[14C]acetylglucosamine gives rise to three different components, including a lipid bound oligosaccharide containing 3- 5 sugar residues.     

Synthesis of UDP-N-[1-14C]acetyl D-glucosamine and UDP-N-[1-14C]acetyl-D-galactosamine from [1-14C]acetate

Procedures for the preparation of UDP-N-[1-14C]acetyl-D-glucosamine and UDP-N-[1-14C]acetyl-D-galactosamine with very high specific activities are described. The overall yield based on the amount of [1-14C]acetate used is greater than 80%. The N-acetyl-D-glucosamine-alpha-1-phosphate used in this synthesis is prepared by phosphorylation of tetraacetyl-D-N-acetylglucosamine with crystalline phosphoric acid. N-acetyl-D-glucosamine-alpha-1-phosphate is then deacetylated in anhydrous hydrazine with hydrazine sulfate as a catalyst. D-glucosamine-alpha-1-phosphate is N-acetylated with [14C]acetate using N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline as the coupling agent. The acetylated product is coverted to the UDP derivative with yeast UDP-N-acetyl-D-glucosamine pyrophosphorylase. UDP-N-[1-14C]acetylgalactosamine is prepared by acetylation of UDP-galactosamine using [1-14C]acetate and N-ethoxy-carbonyl-2-ethoxy-1,2-dihydroquinoline. UDP-galactosamine is prepared enzymatically using galactokinase and galactose-1-phosphate uridyltransferase. The labeled products, isolated and characterized by ion-exchange and paper chromatography, were active as substrates in glycosyl transferase systems.     

Synthesis of acyclo C-nucleosides of phenanthro[9,10-e][1,2, 4]triazino[3,4-c]-[1,2,4]triazoles, and their precursors

Reaction of 3-hydrazinophenanthro [9, 10-e] [1, 2, 4] triazine (1) with aliphatic and aromatic aldehydes as well as monosaccharides gave the corresponding hydrazones 2a-g. The D-glucose analogue exists in the cyclic pyranosyl structure 5. Acetylation and partial acetylation of the sugar hydrazones were carried out. Cyclization of a number of hydrazones including the partially acetylated sugar hydrazones by thionyl chloride gave regioselectively the respective angular isomer 1-substituted phenanthro [9, 10-e] [1, 2, 4] triazino [3, 4- and not the linear isomer. The cyclization of 1 with acetic acid, however, gave regioselectively the linear isomer 19. The structural assignments were based on a model study whereby the angular 16a was found to be different from the linear isomer 19a obtained by the condensation of 4, 5-diamino-3-methyl-1, 2, 4-triazole with 9, 10-phenanthraquinone. Periodate oxidation of 2d gave 20 whose reaction with 1 gave 21.     

Immunohistochemical localization of serotonin transporter in normal and colchicine treated rat brain

The activity of liver microsomal CYP2E1 is commonly measured as the rate of 5-chloro-2-benzoxazolone (chlorzoxazone) 6-hydroxylation, which requires separation of 6-hydroxychlorzoxazone and chlorzoxazone by high pressure liquid chromatography (HPLC). In the present study, we describe a solvent extraction (non-HPLC) assay for measuring CYP2E1 activity, based on the 6-hydroxylation of [14C]chlorzoxazone. When [14C]chlorzoxazone was incubated with human or rat liver microsomes in the presence of NADPH, the major product formed was 6-[14C]hydroxychlorzoxazone. Unreacted [14C]chlorzoxazone was quantitatively extracted from the incubation mixture with dichloromethane under conditions that resulted in approximately 45% extraction of 6-[14C]hydroxychlorzoxazone. The amount of 6-[14C]hydroxychlorzoxazone remaining in the aqueous incubation mixture ( approximately 55% of the total amount formed) was quantified by liquid scintillation spectrometry. The limit of detection for this assay was 100 pmol of 6-[14C]hydroxychlorzoxazone. The solvent extraction procedure was validated by comparing the rates of formation of 6-[14C]hydroxychlorzoxazone with those determined by HPLC under a variety of experimental conditions. The close correspondence between the two analytical methods suggests that the extraction procedure for measuring 6-[14C]hydroxychlorzoxazone provides a simple, sensitive, and rapid alternative to the HPLC procedure for measuring CYP2E1 activity. In rats, the assay is not specific for CYP2E1 because CYP1A1 also catalyzes the 6-hydroxylation of chlorzoxazone. Recombinant human CYP1A1 also catalyzed the 6-hydroxylation of chlorzoxazone (at (1)/(5) the rate of CYP2E1), although CYP1A1 is not expressed in human liver microsomes. The non-HPLC assay was used to investigate the postulated role of CYP1A2 in the 6-hydroxylation of chlorzoxazone by human liver microsomes. Recombinant CYP1A2 did not catalyze the 6-hydroxylation of chlorzoxazone, and studies with 1-[(3,4-dimethoxyphenyl)methyl]-6,7-dimethoxyisoquinoline, which inhibits CYP1A2 but not CYP2E1, indicated that, in human liver microsomes, the 6-hydroxylation of chlorzoxazone is catalyzed by CYP2E1 with little or no contribution from CYP1A2 enzymes over a wide range of substrate concentrations.     

Synthetic peptides related to laminin pentapeptide (YIGSR) fragment

The synthetic laminin pentapeptide amide fragment (LF), Tyr-Ile-Gly-Ser-Arg-NH2 corresponding to a part of B1 chain of the glycoprotein, laminin, and six of its analogues having structural modifications at positions 1, 3 and 4 were synthesized by solid phase method employing mainly 9-fluorenylmethoxycarbonyl-amino acid trichlorophenyl esters as coupling agents and Merrifield resin as the solid support. Their biological activities were studied in vivo by lung tumor colonization assay and in vitro by cell adhesion assay. The activity of synthetic LF was found to correlate with the earlier reported results in both in vivo and in vitro assays. Among the analogues made, [Tyr4] LF and [Thr4]LF were found to inhibit the lung tumor colonies more efficiently than LF itself in the in vivo assay whereas [D- Ser4]LF exhibited almost the same inhibition as LF.     

Effect of squalestatin 1 on the biosynthesis of the mevalonate pathway lipids

The effects of squalestatin 1 on rat brain and liver homogenates and on Chinese hamster ovary tissue culture cells have been investigated. This compound effectively inhibits squalene biosynthesis in a highly selective manner. Cytoplasmic farnesyl pyrophosphate and geranylgeranyl pyrophosphate synthases are not affected, which is also the case for microsomal cis-prenyltransferase. In tissue culture cells, squalestatin 1 inhibits cholesterol biosynthesis completely, but does not alter dolichol synthesis or protein isoprenylation to a great extent. Incorporation of [3H]mevalonate into ubiquinone-9 and -10 increases 3-4-fold, probably as a result of increased synthesis of this lipid. Squalestatin 1 appears not only to be an effective inhibitor of cholesterol biosynthesis, but also to be more specific than other inhibitors used earlier in various in vitro and in vivo systems.     

Kinetic characterization of the interaction of biotinylated human interleukin 5 with an Fc chimera of its receptor alpha subunit and development of an ELISA screening assay using real-time interaction biosensor analysis

The interaction of biotinylated human interleukin 5 ([BT]hIL5) with immobilized receptor was measured with a real-time biosensor, and these results were used as a basis for configuring an ELISA for screening antagonists of hIL5-receptor binding. The recombinant proteins used, hIL5 and shIL5R alpha-Fc (chimeric fusion receptor constructed by linking the soluble component of the hIL5 receptor alpha subunit to the constant domain (Fc) of immunoglobulin G), were produced by the expression of cloned vectors in Drosophila schneider (S2) cells. Initial attempts to develop a screening assay by direct immobilization of soluble IL5 receptor to microtiter plates proved unsatisfactory and led to use of the Fc chimera attached by oriented immobilization via protein A. Hence, shIL5R alpha-Fc was bound to protein A covalently immobilized on a carboxymethyl dextran (CM-5) biosensor chip. Specific binding was demonstrated of [BT]hIL5 to protein A/shIL5R alpha-Fc receptor complex. The binding was high affinity (Kdapp = 6 nM), reversible and saturable. The affinity of [BT]hIL5 was similar to that determined with the biosensor assay for unmodified hIL5. The observed kinetics of the interactions of Fc chimera with protein A (slow dissociation) and of [BT]hIL5 with immobilized Fc chimera (faster dissociation) were favorable for subsequently establishing a microtiter plate based ELISA assay. In the latter, Fc chimera was immobilized to the plate via protein A as in the biosensor experiment. Binding of [BT]hIL5 to immobilized Fc chimera in the ELISA was concentration dependent and was competed by both hIL5 and shIL5R alpha.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of binding properties to human P-glycoprotein: development of a [3H]verapamil radioligand-binding assay

Interaction with the exsorptive transporter P-glycoprotein (P-gp) is a possible source of peculiarities in drug pharmacokinetics, including dose-dependent absorption, drug-drug interactions, intestinal secretion, and limited permeability of the blood-brain barrier. Among the established in vitro methods of the analysis of drug interactions with P-gp, none directly quantifies the affinity of ligands with P-gp. Instead, they measure the result of a membrane permeation and a receptor-binding process; this may lead to difficulties in the interpretation of results. An assay for quantification of drug affinity to the transporter is presented on the basis of the radioligand-binding assay principle. This has the advantage of directly quantifying the interaction between drugs and P-gp. Because of the reversible and competitive interaction of numerous substrates with P-gp, a radioligand-binding assay was developed by taking [3H]verapamil and [3H]vinblastine as radioligands and the human intestinal Caco-2 cells, overexpressed with P-gp by culturing in the presence of vinblastine or transfecting with multidrug resistance gene MDR-1 as receptor preparation. The assay was performed in 96-well plates and has the potential to be used as a high-throughput method. A clear induction of the expression of P-gp was demonstrated in the Caco-2 cells grown in the presence of vinblastine, as well as in the transfected cells, although to a lesser extent. Both radioligands were shown to bind to P-gp. Verapamil was the radioligand of choice for further investigations due to its lower nonspecific binding to the transporter preparation. Kinetics as well as specificity of the binding of verapamil to the P-gp preparation were demonstrated. A two-affinity model was found to adequately describe the data derived from saturation as well as from competition experiments, in accordance with previous findings on two exsorption sites for P-gp. The binding properties of [3H]verapamil and [3H]vinblastine to a P-gp preparation derived from induced Caco-2 cells are described. The concentration-dependent displacement of the radioligand by nonlabeled substrates for P-gp should be a suitable principle for the determination of drug affinity to the respective binding sites at the human intestinal multidrug transporter P-gp.     

Improvement in nerve regeneration by monoclonal antibodies to ICAM-1 and LFA-1 in allogeneic mice

A mixed membrane fraction isolated from C. albicans yeast cells catalyzed the transfer of glucose from UDP-Glc into three classes of endogenous acceptors: glucolipid, glycoprotein and lipid-linked oligosaccharides. About 80% of the total radioactivity transferred into these products corresponded to the glucolipid which was identified as dolichol phosphate glucose by several criteria. The remainder was detected in about equal proportions in the other two fractions. Conditions that stimulated or inhibited glucolipid synthesis did not affect the extent of glycoprotein labeling. The synthesis of dolichol phosphate glucose exhibited a K(m) of 104 microM UDP-Glc and was stimulated by Mg2+ but not by Mn2+ or Ca2+. The latter cations were, however, better stimulators of glycoprotein labeling than Mg2+. Most nucleotides strongly inhibited the synthesis of dolichol phosphate glucose, UMP being a competitive inhibitor with a Ki of 100 microM. The dolichol phosphate glucose synthase reaction was reversed about 57% by 0.62 mM UDP but not by UMP.     

Characterization of a 2[4Fe-4S] ferredoxin obtained by chemical insertion of the Fe-S clusters into the apoferredoxin II from Rhodobacter capsulatus

The Rhodobacter capsulatus ferredoxin II (FdII) belongs to a family of 7Fe ferredoxins containing one [3Fe-4S] cluster and one [4Fe-4S] cluster. This protein, encoded by the fdxA gene, has been overproduced in Escherichia coli as a soluble apoferredoxin. The purified recombinant protein was subjected to reconstitution experiments by chemical incorporation of the Fe-S clusters under anaerobic conditions. A brown protein was obtained, the formation of which was dependent upon the complete unfolding of the polypeptide prior to incorporation of iron and sulfur atoms. The yield of the reconstituted product was higher when the reaction was carried out at slightly basic pH. The reconstituted ferredoxin was purified and shown to be distinct from the native [7Fe-8S] ferredoxin, based on several biochemical and spectroscopic criteria. In the oxidized state, EPR revealed the quasi-absence of [3Fe-4S] cluster. 1H-NMR spectroscopic analyses provided evidence that the protein was reconstituted as a 2[4Fe-4S] ferredoxin. This conclusion was further supported by the determination by electrospray mass spectrometry of the molecular mass of the reconstituted protein, which matched within 2 Da to the mass of the FdII polypeptide incremented of eight atoms each of iron and sulfur. Exposure of the reconstituted protein to air resulted in a fast and irreversible oxidative denaturation of the Fe-S clusters, without formation of [7Fe-8S] form. Unlike the natural 7Fe ferredoxin, the reconstituted ferredoxin appeared incompetent in an electron-transfer assay coupled to nitrogenase activity. The fact that the apoFdII was reconstituted as a highly unstable 8Fe ferredoxin instead of the 7Fe naturally occurring FdII is discussed in relation to the results obtained with other types of ferredoxins.     

Metabolism of [1-(14)C] and [2-(14)C] leucine in cultured skin fibroblasts from patients with isovaleric acidemia. Characterization of metabolic defects

Leucine metabolism in cultured skin fibroblasts from patients with isovaleric acidemia was compared with that in normal fibroblasts and in cells from patients with maple syrup urine disease using [1-(14)C] and [2-(14)C] leucine as substrates. Inhibitory effects of methylenecyclopropylacetic acid on leucine metabolism in normal cells were also investigated. Production of 14CO2 from [2-(14)C] leucine was very reduced (96-99%) in both types of mutant cells. Radioactive isovaleric acid accumulated in assay media with isovaleric acidemia cells but not in those with maple syrup urine disease cells. Unexpectedly, 14CO2 production from [1-(14)C] leucine was partially depressed (80%) in isovaleric acidemia cells whereas in maple syrup urine disease cells it was strongly depressed (99%) as expected. These two mutant cells were clearly distinguished by detection of 14C-isovaleric acid accumulation after incubation with [2-(14)C] leucine. A pattern of inhibition of leucine oxidation similar to that seen in isovaleric acidemia cells was induced in normal cells by the addition of 0.7 mM methylenecyclopropylacetic acid to the assay medium. The partial inhibition of [1-(14)C] leucine oxidation seen in isovaleric acidemia cells and also in normal cells in the presence of the inhibitor appears to be, at least in part, due to an accumulation of isovalerate in the cells. Isovaleric acid (5-10) mM) inhibited [1-(14)C] leucine oxidation 32-68% when added to the assay medium with normal cells. Addition of flavin adenine dinucleoside to culture medium or assay medium or both did not restore oxidation of either leucine substrate in isovaleric acidemia cells.     

Nonpeptide peptidomimetic antagonists of the neuropeptide Y receptor: benextramine analogs with selectivity for the peripheral Y2 receptor

We synthesized a new series of benextramine analogs as neuropeptide Y (NPY) functional group mimetics and tested them for N-[propionyl-3H]NPY ([3]NPY) displacement activity in rat brain membrane homogenates and for NPY receptor antagonist activity in the rat femoral artery. The tetraamine, carbon analog N,N'-bis[6-[N-(2-naphthylmethyl)amino]hexyl]-1,6-hexanediamine (15) was equipotent with benextramine (based on comparison of the relevant IC50's) in a rat brain [3H]NPY displacement assay, suggesting that the disulfide is not a necessary feature of benextramine's [3H]NPY displacement activity, although this analog maintained selectivity for the benextramine-sensitive binding site population. The bis(N,N-dialkylguanyl) disulfide and carbon analogs 14a-c were 3-4 times more potent than their respective controls in displacing [3H]NPY from rat brain membrane homogenates with IC50's ranging from 15 to 18 microM and maintained selectivity for the benextramine-sensitive, Y1 binding site population. However, the activity of the carbon analog N,N'-bis[6-[N-(2-naphthylmethyl)amino]hexyl]-N,N'-(1,6- hexanediyl)diguanidine tetrahydrochloride (14b) showed a different profile in a femoral artery vasoconstriction assay; at 1.0 nM, this analog shifted the concentration-effect curve of the Y2-selective agonist NPY13-36 to the right (pA2 = 9.2; Kd = 0.63 nM) without a significant change in the maximum effect, while even at 1.0 mM it had no effect on the vasoconstrictive activity of the Y1-selective agonist [Leu31,Pro34]NPY. Thus, the bis(N,N-dialkylguanidine) analogs of benextramine are selective, competitive antagonists of the postsynaptic NPY receptor in the femoral artery.     

Multibranched V3 peptides inhibit human immunodeficiency virus infection in human lymphocytes and macrophages

Synthetic polymeric constructions (SPCs) including the consensus sequence of the human immunodeficiency virus type 1 (HIV-1) surface envelope glycoprotein gp120 V3 loop (GPGRAF) blocked the fusion between HIV-1- and HIV-2-infected cells and CD4+ uninfected cells. A structure-activity relationship study using V3 SPC analogs showed that the most efficient inhibitor of cell fusion was an eight-branched SPC with the hexapeptide motif GPGRAF (i.e., [GPGRAF]8-SPC). N-terminal acetylation or incorporation of D-amino acids in the GPGRAF sequence of this SPC resulted in significant loss of activity. Analogs with fewer than six residues in the motif (i.e., GPGRA or GPGR), as well as SPCs with a nonrelevant sequence, did not inhibit cell fusion, demonstrating the high specificity of the antifusion activity. [GPGRAF]8-SPC, which was not toxic to CEM cells at concentrations of up to 50 microM, inhibited 50% of HIV-1(LAI) replication in these cells at a concentration of 0.07 microM. Moreover, [GPGRAF]8-SPC inhibited the infection of human peripheral blood mononuclear cells by several HIV-1 and HIV-2 isolates, including laboratory strains [HIV-1(LAI), HIV-1(NDK), and HIV-2(ROD)], and fresh primary isolates, including two zidovudine-resistant HIV-1 isolates and two HIV-2 isolates obtained from infected individuals. The multibranched peptide also inhibited infection of human primary macrophages by the highly cytopathic macrophage-tropic isolate HIV-1(89.6). The antiviral activity of [GPGRAF]8-SPC was not related to a virucidal effect, since preincubation of HIV-1 with the peptide did not affect its infectious titer. This result is in agreement with the concept that the multibranched peptide mimics a part of the V3 loop and thus interacts with the host cell. The therapeutic properties of synthetic multibranched peptides based on the V3 loop consensus motif should be evaluated in HIV-infected patients.     

A randomized biomechanical study of zone II human flexor tendon repairs analyzed in an in vitro model

Human telomerase, a ribonucleoprotein enzyme is known to be associated with immortalized cancer cells but is absent in most normal tissues. Thus, telomerase appears to be an attractive new target for anticancer agents and an important diagnostic marker of human cancers. Here, we describe an improved telomerase assay method based on the Dynabead biomagnetic separation theory. In this method, 5'-biotinylated (TTAGGG)3 was used as a primer for the telomerase reaction. Telomerase reaction products were then immobilized on streptavidin-coated Dynabeads and washed intensively to eliminate excess [alpha-32P]dGTP. Using this method, without the amplification of telomerase reaction products by the PCR, we were able to quantitatively detect telomerase activity in human HeLa cell extracts equivalent to between 200-500 cells. This method is anticipated to be useful for the measurement of telomerase activity in various tumor cells, for assessing potential telomerase and for understanding the biochemical aspects of the telomerase reaction.     

Insulin utilization and kinetic effect on hybridoma metabolism in batch and continuous cultures

The neuropeptide growth factor antagonists [D-Arg1,D-Phe5,D-Trp7,9,Leu11]-substance P (D) and [Arg6,D-Trp7,9, [corrected] N-MePhe8]-substance P(6-11) (G) are currently undergoing preclinical evaluation as potential anticancer agents and clinical trials are planned for G in the near future. A reversed-phase high-performance liquid chromatographic separation has been developed which is both sensitive (limit of detection 250 pg/263 fmol for G; 500 pg/330 fmol for D) and selective, based on electrochemical detection of the two tryptophan residues present in each peptide. Two ion-pairing agents were included in the isocratic mobile phase to eliminate adsorption of the peptides onto the analytical column. Extensive sample clean-up procedures have been developed for plasma, tissue and tumour based on solid-phase extraction. Precision and accuracy of each assay was 91.3 +/- 16.9% (between-day) for G and 99.3 +/- 16.9% (between-day) for D. The assays were able to detect the intact peptides and a number of their metabolites in plasma, liver and the WX 322 SCLC human xenograft in nude mice for at least 6 hr after administration of therapeutic and pharmacological doses.     

A detergent-solubilized nicotinic acetylcholine receptor of Caenorhabditis elegans

We have used a spin column assay to study the detergent-solubilized levamisole receptor, a nicotinic acetylcholine receptor of the nematode Caenorhabditis elegans. The receptor can be successfully solubilized in detergent solutions of Triton X-100, Lubrol PX, or sodium cholate. Centrifugal gel filtration assay using the tritiated ligand [3H]meta-aminolevamisole ([3H]MAL) provides a greater signal and a better signal-to-noise ratio for soluble levamisole receptor binding than either polyethylene glycol precipitation or DEAE filter assay with the same ligand. As for membrane-bound receptor, the detergent-solubilized levamisole receptor consists of more than one affinity state. Detergent solubilization appears to increase the affinity of all states for [3H]MAL (Kd for the highest affinity solubilized [3H]MAL binding state, 41 +/- 5 pM). Data is presented on the equilibrium binding and the association and dissociation reaction rates of the receptor. The similar relative efficacy with which various compounds inhibit specific [3H]MAL binding and deficiencies in solubilizable high affinity specific [3H]MAL binding in two receptor mutants show that the solubilized receptor is the same nicotinic acetylcholine receptor that is detected by assaying membrane-bound specific [3H]MAL binding. The detergent-solubilized levamisole receptor is stable at 0 degree to 4 degrees C, making receptor purification feasible.     

Renal blood flow velocity in acute renal failure following cardiopulmonary bypass surgery

Two lysosomal storage diseases, aspartylglucosaminuria and mannosidosis, are associated with highly elevated serum dolichol concentrations. To elucidate possible mechanisms leading to elevated serum dolichols, we studied the effects of Triton WR 1339 (known to increase serum cholesterol) and orotic acid (known to decrease serum cholesterol) on blood and biliary dolichol and beta-hexosaminidase levels in rats. In Triton WR 1339-treated rats, serum dolichol was markedly increased compared with saline-treated controls 1 (400 +/- 70 ng/mL, n = 7 v 85 +/- 11 ng/mL, n = 8, P < .001), 4 (789 +/- 70 ng/mL, n = 10 v 110 +/- 10 ng/mL, n = 7, P < .0001), and 8 (549 +/- 43 ng/mL, n = 8 v 87 +/- 8 ng/mL, n = 7, P < .001) days after administration of the drug. By contrast, serum dolichol was decreased (64 +/- 5 ng/mL, n = 8 v 119 +/- 7 ng/mL, n = 8, P < .0001) after a 7-day orotic acid feeding compared with controls. Serum beta-hexosaminidase was unaffected by both treatments. Orotic acid also increased biliary dolichol (280 +/- 47 ng/100 g body weight [BW]/h, n = 7 v 83 +/- 15 ng/100 g BW/h, n = 7, P < .01) and beta-hexosaminidase (21 +/- 3 mU/100 g BW/h, n = 7 v 8.3 +/- 2 mU/100 g BW/h, n = 9, P < .01) excretion compared with controls. Thus, both Triton WR 1339 and orotic acid have an effect on dolichol metabolism, and it is conceivable--based on our results--that serum dolichol concentrations are regulated, at least in part, by a mechanism similar to that for serum cholesterol levels.     

Gas chromatographic-mass spectrometric determination of benzo[a]pyrene and chrysene diol epoxide globin adducts in humans

The efficacy of a newly developed gas chromatography-negative ion chemical ionization-mass spectrometry-selected ion monitoring (GC-NICI-MS-SIM) assay for measuring globin adducts of benzo[a]pyrene (B[a]P) and chrysene diol epoxides in human was evaluated. In this pilot study, smokers and nonsmokers were selected as exposed and nonexposed groups. Using [2H12]r-7,t-8,9,c-10-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyren e ([2H12]trans,anti-B[a]P-tetraol) as an internal standard, B[a]P-tetraols released from globin after hydrolysis and derivatization were quantified by GC-NICI-MS-SIM. Levels of trans-1,2-dihydroxy-3,4-epoxy-1,2,3,4-tetrahydrochrysene (chrysene-DE)-globin adducts were estimated by assuming that the recovery and the MS response of the perdeuterated B[a]P-tetraol internal standard reflected the recovery and MS response of chrysene tetraols. The assay was found to be reproducible and sensitive enough to detect both analytes in all samples. The mean levels of B[a]P-tetraols released from the corresponding benzo[a]pyrene diol epoxide (BPDE) globin adducts in smokers were significantly higher than those in nonsmokers, i.e., 2.6 +/- 0.6 SE fmol/mg globin (ranging from 1.2 to 7.8 fmol/mg globin) in smokers and 0.97 +/- 0.05 SE fmol/mg globin (ranging from 0.7 to 1.3 fmol/mg globin) in nonsmokers (P < 0.01). Interestingly, estimated levels of chrysene-DE-globin adducts in the same subjects were about two orders of magnitude higher than those of the globin adducts of BPDE. The mean of the chrysene-DE adducts in smokers was estimated to be 310 +/- 30 SE fmol/mg globin (ranging from 190 to 460 fmol/mg globin) and that in nonsmokers was 250 +/- 25 SE fmol/mg globin (ranging from 110 to 380 fmol/mg). Although the estimated mean of chrysene-DE adducts with globin in smokers appeared to be about 25% higher than in nonsmokers, the difference was not significant (P = 0.06). The results of this study demonstrate the feasibility of the GC-NICI-MS-SIM method for measurement of BPDE globin adducts in humans.