Selective identification of cyclopentaring-fused PAHs and side-substituted PAHs in a low pressure premixed sooting flame by photoelectron photoion coincidence spectroscopy.

This work reports on the selective on-line identification of polycyclic aromatic hydrocarbons (PAHs) formed in a low-pressure methane sooting flame, carried out using the double imaging Photoelectron Photoion Coincidence Spectroscopy method (i2PEPICO) on the DESIRS VUV beamline at the synchrotron SOLEIL. Generally, this work demonstrates the capabilities of the i2PEPICO method to identify PAHs in sooting flames, and in particular to distinguish cyclopentaring-fused PAHs (CP-PAHs) and side-substituted PAHs from their benzenoid isomers. Experimental threshold photoelectron spectra of four CP-PAHs: acenapthylene (C12H8, 152 m/z), acenaphtene (C12H10, 154 m/z), fluoranthene (C16H10, 202 m/z) and benzo(ghi)fluoranthene (C18H10, 226 m/z) are also reported for the first time.

Pharmacokinetic and safety study of subcutaneously administered weekly ING-1, a human engineere monoclonal antibody targeting human EpCAM, in patients with advanced solid tumors.

BACKGROUND: ING-1 is a high-affinity, human engineeredtrade mark monoclonal antibody that recognizes a 40 kilodalton epithelial cell adhesion molecule (EpCAM) glycoprotein that is expressed in high levels on most adenocarcinomas and is an attractive target for immunotherapy. METHODS: ING-1 was administered subcutaneously weekly at doses between 0.1 and 2 mg/kg/week. Pharmacokinetic samples were drawn during weeks 1 and 6. RESULTS: Fourteen patients with advanced refractory cancer received a median of 6 (range 1-9) doses of ING-1. At 1 mg/kg, a 62-year-old man with colon cancer developed reversible grade 3 pancreatitis after the third dose. His plasma ING-1 levels were similar to the other two patients dosed at 1 mg/kg. Two patients dosed at 0.6 mg/kg experienced stable disease at 6 weeks. Peak drug levels increased with dose and time, suggesting drug accumulation with repeated dosing. Low human anti-human antibody response was noted in three of the 13 patients assessed and was directed towards the variable region of ING-1. CONCLUSIONS: Weekly ING-1 administered subcutaneously was well tolerated at 0.6 mg/kg/week and further experience at this dose is warranted to demonstrate safety. The risk of pancreatitis and the marginal anti-tumor effect may preclude further monotherapy studies; however, combination studies with chemotherapy are warranted.

Characterization of aqueous and solid inclusion complexes of diuron and isoproturon with beta-cyclodextrin.

The interaction of diuron and isoproturon herbicides with beta-cyclodextrin is conducive to the formation of inclusion compounds in aqueous solution as well as in the solid state. The physico-chemical study of these complexes was carried out by various analytical techniques such as ultraviolet (UV), Fourier transform infrared (FT-IR), Raman, X-ray diffraction, and 1H-NMR (nuclear magnetic resonance) spectroscopies. The existence of inclusion complexes in water solution between the beta-cyclodextrin and each of the herbicides was revealed by electronic absorption and 1H-NMR spectroscopies. A 1:1 stoichiometry was determined for both complexes in aqueous medium from UV absorption spectra by using the Benesi-Hildebrand method; the relative stability constants at room temperature were calculated at 2700 +/- 300 L mol(-1) and 750 +/- 50 L mol(-1) for isoproturon and diuron, respectively. In the solid state, inclusion processes with beta-cyclodextrin were characterized by means of infrared and Raman techniques and confirmed by X-ray diffraction spectra.

Pharmacokinetics of saquinavir, zidovudine, and zalcitabine in combination therapy.

We investigated the pharmacokinetics of zidovudine, zalcitabine, and saquinavir in AIDS Clinical Trial Group protocol 229. Patients received either saquinavir, zalcitabine, or a combination of both, together with zidovudine three times a day. Approximately 100 patients were enrolled in each treatment arm, and intensive pharmacokinetic studies were performed on about 25 patients per arm at weeks 1 and 12. We estimated the pharmacokinetic parameters of all three drugs by using parametric and nonparametric methods. The mean values of the pharmacokinetic parameters of zidovudine (clearance [CL]/bioavailability [F], 168 liters/h; volume of distribution [V]/F, 185 liters; half-life, 0.76 h) and zalcitabine (CL/F, 25 liters/h; V/F, 92.2 liters; half-life, 2.7 h) were similar to those reported previously. For saquinavir, the mean pharmacokinetic parameter estimates using parametric methods were as follows: maximum concentration of drug in serum [Cmax], 70.8 ng/ml; time to Cmax, 3.11 h; area under the curve, 809 ng x h/ml; CL/F, 989 liters/h; V/F, 1,503 liters; half-life, 1.38 h. For all three drugs, clearance decreased with age. Weight did not influence the clearance of zidovudine, but the clearance of zalcitabine and saquinavir increased with weight. There were no differences in pharmacokinetic parameters between study weeks and arms, suggesting that there is no change in kinetics with chronic administration and that there are no significant pharmacokinetic interactions among these three drugs.

Exposure-response relationships for saquinavir, zidovudine, and zalcitabine in combination therapy.

The relationship of CD4+ cell response, level of RNA in plasma, and quantitative peripheral blood mononuclear cell (PBMC) titer to apparent drug exposure was investigated by using data from AIDS Clinical Trial Group protocol 229, a multicenter randomized study. Patients received either saquinavir, zalcitabine, or a combination of both, along with open-label zidovudine. Approximately 100 patients were enrolled in each arm, and the primary study duration was 24 weeks. Individual drug exposure, the area under the concentration-time curve, was estimated by using population-based pharmacokinetic methods. Response was defined as the maximum increase in CD4+ cell count or the maximum decrease in RNA in plasma or PBMC titer adjusted for baseline CD4+ cell count, RNA in plasma, and PBMC titer, respectively. Regression of responses on exposure demonstrated an exposure effect for saquinavir which was significant for the maximum increase in CD4+ cell count and the decrease in RNA in plasma. For the PBMC titer, no significant relationship could be demonstrated but the results suggested a trend similar to that of the other response variables. For all three response variables, the slope of the saquinavir exposure response was greater with the triple combination (saquinavir, zidovudine, and zalcitabine) than with the combination of saquinavir and zidovudine, suggesting possible synergism between saquinavir and zalcitabine.

Peroxisomal beta-oxidation. Purification of four novel 3-hydroxyacyl-CoA dehydrogenases from rat liver peroxisomes.

Peroxisomes are capable of beta-oxidizing a variety of substrates including the CoA esters of straight chain fatty acids, 2-methyl-branched fatty acids and the bile acid intermediates di- and trihydroxycoprostanic acids. The first reaction of peroxisomal beta-oxidation is catalyzed by an acyl-CoA oxidase. Rat liver peroxisomes contain three acyl-CoA oxidases: 1) palmitoyl-CoA oxidase, oxidizing straight chain acyl-CoAs; 2) pristanoyl-CoA oxidase, oxidizing 2-methyl-branched acyl-CoAs; and 3) trihydroxycoprostanoyl-CoA oxidase, oxidizing the CoA esters of the bile acid intermediates (Van Veldhoven, P.P., Vanhove, G., Asselberghs, S., Eyssen, H. J., and Mannaerts, G. P. (1992) J. Biol. Chem. 267, 20065-20074). We have now investigated whether the third step of peroxisomal beta-oxidation, catalyzed by a 3-hydroxyacyl-CoA dehydrogenase, is also catalyzed by multiple enzymes, using the 3-hydroxyacyl-CoA derivatives of palmitic acid, 2-methylpalmitic acid, and trihydroxycoprostanic acid as the substrates to monitor the dehydrogenase activities. In order to avoid contamination with mitochondrial 3-hydroxyacyl-CoA dehydrogenases, highly purified peroxisomes from untreated rats were employed as the enzyme source. Subfractionation of the peroxisomes revealed that the major portion of the dehydrogenase activities with all three substrates was present in the peripheral membrane protein fraction. Separation of this fraction on various chromatographic columns resulted in the purification of the well known multifunctional protein, a 78-kDa monomeric protein that displays 3-hydroxyacyl-CoA dehydrogenase plus hydratase activity, as well as of four additional novel dehydrogenases with different substrate specificities. Three of the enzymes are monomeric proteins of 35 kDa, 56 kDa, and 79 kDa, respectively. The latter enzyme also displays hydratase activity. The fourth enzyme is a dimer of 89 kDa, the subunits of which form a doublet at 40 kDa. The exact physiological role of each of the 3-hydroxyacyl-CoA dehydrogenases requires further investigation.

A new peroxisomal disorder with fetal and neonatal adrenal insufficiency.

A boy with a new type of adrenoleukodystrophy is described. This was characterised by fetal and neonatal adrenal insufficiency, a neurological picture as seen in neonatal adrenoleukodystrophy, but with a normal number of peroxisomes in the liver and a peroxisomal dysfunction limited to the very long chain fatty acids and pristanic acid.

The CoA esters of 2-methyl-branched chain fatty acids and of the bile acid intermediates di- and trihydroxycoprostanic acids are oxidized by one single peroxisomal branched chain acyl-CoA oxidase in human liver and kidney.

Rat liver peroxisomes contain three acyl-CoA oxidases: palmitoyl-CoA oxidase, which oxidizes the CoA esters of straight chain fatty acids and prostaglandins; pristanoyl-CoA oxidase, which oxidizes the CoA esters of 2-methyl-branched fatty acids (e.g. pristanic acid); and trihydroxycoprostanoyl-CoA oxidase, which oxidizes the CoA esters of the bile acid intermediates di- and trihydroxycoprostanic acids (Van Veldhoven, P. P., Vanhove, G., Asselberghs, S., Eyssen, H. J., and Mannaerts, G. P. (1992) J. Biol. Chem. 267, 20065-20074). In the present report we demonstrate that human liver peroxisomes contain only two acyl-CoA oxidases: palmitoyl-CoA oxidase, which oxidizes the CoA esters of straight chain fatty acids and prostaglandins, and a novel branched chain acyl-CoA oxidase, which oxidizes the CoA esters of 2-methyl-branched fatty acids as well as those of the bile acid intermediates (which also possess a 2-methyl substitution in their side chains). The branched chain acyl-CoA oxidase was purified to near homogeneity by means of column chromatography. It appeared to be a 70-kDa monomeric protein that did not cross-react with antisera raised against rat palmitoyl-CoA oxidase and pristanoyl-CoA oxidase. No indication was found for the presence of a separate trihydroxycoprostanoyl-CoA oxidase in human liver. The branched chain acyl-CoA oxidase was present also in human kidney, suggesting that it is expressed in other extrahepatic tissues as well. Our results explain a number of clinical-chemical observations made in certain cases of peroxisomal beta-oxidation disorders.

Mitochondrial short-chain acyl-CoA dehydrogenase of human liver and kidney can function as an oxidase.

During an attempt to purify the peroxisomal acyl-CoA oxidases from human liver and kidney, we discovered a novel short-chain acyl-CoA oxidase, which was well separated from the known peroxisomal oxidases on various chromatographic columns. However, further experiments demonstrated that the novel oxidase is identical with the mitochondrial short-chain acyl-CoA dehydrogenase. (1) Subcellular fractionation revealed that the short-chain acyl-CoA oxidase is present in mitochondria and absent from peroxisomes. (2) The molecular mass (43 kDa) of the subunit of the purified oxidase was similar to that reported for the dehydrogenase. (3) The substrate spectrum of the oxidase was comparable with that described for the dehydrogenase. (4) On column chromatography, the oxidase and dehydrogenase activities co-eluted. Our results indicate that, in the absence of suitable electron acceptors, the short-chain acyl-CoA dehydrogenase is capable of transferring electrons directly to molecular oxygen, yielding potentially harmful H2O2. This raises the question as to whether the dehydrogenase might function as an oxidase in conditions in which the activity of the electron-transport chain is decreased, such as reperfusion after ischaemia.

Substrate specificities of rat liver peroxisomal acyl-CoA oxidases: palmitoyl-CoA oxidase (inducible acyl-CoA oxidase), pristanoyl-CoA oxidase (non-inducible acyl-CoA oxidase), and trihydroxycoprostanoyl-CoA oxidase.

Rat liver peroxisomes contain three acyl-CoA oxidases:palmitoyl-CoA oxidase, pristanoyl-CoA oxidase, and trihydroxycoprostanoyl-CoA oxidase. The three oxidases were separated by anion-exchange chromatography of a partially purified oxidase preparation, and the column eluate was analyzed for oxidase activity with different acyl-CoAs. Short chain mono (hexanoyl-) and dicarboxylyl (glutaryl-)-CoAs and prostaglandin E2-CoA were oxidized exclusively by palmitoyl-CoA oxidase. Long chain mono (palmitoyl-) and dicarboxylyl (hexadecanedioyl-)-CoAs were oxidized by palmitoyl-CoA oxidase and pristanoyl-CoA oxidase, the former enzyme catalyzing approximately 70% of the total eluate activity. The very long chain lignoceroyl-CoA was also oxidized by palmitoyl-CoA oxidase and pristanoyl-CoA oxidase, the latter enzyme catalyzing approximately 65% of the total eluate activity. Long chain 2-methyl branched acyl-CoAs (2-methylpalmitoyl-CoA and pristanoyl-CoA) were oxidized for approximately 90% by pristanoyl-CoA oxidase, the remaining activity being catalyzed by trihydroxycoprostanoyl-CoA oxidase. The short chain 2-methylhexanoyl-CoA was oxidized by trihydroxycoprostanoyl-CoA oxidase and pristanoyl-CoA oxidase (approximately 60 and 40%, respectively, of the total eluate activity). Trihydroxycoprostanoyl-CoA was oxidized exclusively by trihydroxycoprostanoyl-CoA oxidase. No oxidase activity was found with isovaleryl-CoA and isobutyryl-CoA. Substrate dependences of palmitoyl-CoA oxidase and pristanoyl-CoA oxidase were very similar when assayed with the same (common) substrate. Since the two oxidases were purified to a similar extent and with a similar yield, the contribution of each enzyme to substrate oxidation in the column eluate probably reflects its contribution in the intact liver.

Mitochondrial and peroxisomal beta oxidation of the branched chain fatty acid 2-methylpalmitate in rat liver.

A number of isoprenoids (e.g. pristanic acid and the side chains of fat soluble-vitamins) is degraded or shortened via beta oxidation. We synthesized 2-methyl-palmitate and 2-methyl[1-14C] palmitate as a model substrate for the study of the beta oxidation of branched (isoprenoid) fatty acids in rat liver. 2-Methylpalmitate was well oxidized by isolated hepatocytes and its oxidation was stimulated after treatment of the animals with a peroxisome proliferator. Subcellular fractionation of rat liver demonstrated that 2-methylpalmitate is activated to its CoA ester in endoplasmic reticulum, mitochondria, and peroxisomes and that mitochondria and peroxisomes are capable of beta-oxidizing 2-methylpalmitate. At low unbound 2-methylpalmitate concentrations and in the presence of competing straight chain fatty acids, a condition encountered in vivo, peroxisomal 2-methyl-palmitate oxidation was 2- to 4-fold more active than mitochondrial oxidation. Treatment of rats with a peroxisome proliferator markedly stimulated mitochondrial but only slightly peroxisomal 2-methylpalmitate oxidation. The same treatment dramatically induced palmitoyl-CoA oxidase but did not change 2-methyl-palmitoyl-CoA oxidase activity. Our results indicate 1) that in untreated rats peroxisomes contribute for an important part to the oxidation of 2-methylpalmitate; 2) that treatment with a peroxisome proliferator stimulates mainly the mitochondrial component of 2-methylpalmitate oxidation; and 3) that palmitoyl-CoA and 2-methylpalmitoyl-CoA are oxidized by different peroxisomal oxidases.

Identification and purification of a peroxisomal branched chain fatty acyl-CoA oxidase.

Isoprenoid (branched) fatty acids such as pristanic acid can be degraded via beta-oxidation in peroxisomes. We synthesized 2-methylpalmitoyl-CoA as a model substrate in order to study the first step of the peroxisomal beta-oxidation of branched fatty acids, catalyzed by an acyl-CoA oxidase. 2-Methylpalmitoyl-CoA oxidase activity was found in rat liver homogenates. Subcellular fractionation demonstrated that the oxidase was confined to peroxisomes. 2-Methylpalmitoyl-CoA oxidase was also present in kidney and intestine. It was not induced in liver or in the extrahepatic tissues by treatment of rats with peroxisome proliferators or by feeding diets containing excess isoprenoids. The enzyme was partially purified together with palmitoyl-CoA oxidase and trihydroxycoprostanoyl-CoA oxidase by heat treatment and ammonium sulfate fractionation of liver extracts. The partially purified preparation was chromatographed on various columns. 2-Methylpalmitoyl-CoA oxidase could be separated from the inducible (by peroxisome proliferators) palmitoyl-CoA oxidase and from trihydroxycoprostanoyl-CoA oxidase, but it always coeluted with the noninducible palmitoyl-CoA oxidase, recently described by us (Schepers, L., Van Veldhoven, P. P., Casteels, M., Eyssen, H. J., and Mannaerts, G. P. (1990) J. Biol. Chem. 265, 5242-5246). 2-Methylpalmitoyl-CoA oxidase was purified to near homogeneity in three chromatographic steps (anion exchange, hydroxylapatite, and gel filtration). Its apparent molecular mass is approximately 415 kDa, and it consists of identical subunits of approximately 70 kDa. The enzyme oxidized 2-methylpalmitoyl-CoA twice as rapidly as palmitoyl-CoA and pristanoyl-CoA as rapidly as palmitoyl-CoA, so that it can be considered as a branched fatty acyl-CoA oxidase. Since pristanoyl-CoA is one of its naturally occurring substrates we propose to name this enzyme pristanoyl-CoA oxidase.