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BACKGROUND AND PURPOSE: The percentage signal recovery in non-leakage-corrected (no preload, high flip angle, intermediate TE) DSC-MR imaging is known to differ significantly for glioblastoma, metastasis, and primary CNS lymphoma. Because the percentage signal recovery is influenced by preload and pulse sequence parameters, we investigated whether the percentage signal recovery can still differentiate these common contrast-enhancing neoplasms using a DSC-MR imaging protocol designed for relative CBV accuracy (preload, intermediate flip angle, low TE). MATERIALS AND METHODS: We retrospectively analyzed DSC-MR imaging of treatment-naïve, pathology-proved glioblastomas (n = 14), primary central nervous system lymphomas (n = 7), metastases (n = 20), and meningiomas (n = 13) using a protocol designed for relative CBV accuracy (a one-quarter-dose preload and single-dose bolus of gadobutrol, TR/TE = 1290/40 ms, flip angle = 60° at 1.5T). Mean percentage signal recovery, relative CBV, and normalized baseline signal intensity were compared within contrast-enhancing lesion volumes. Classification accuracy was determined by receiver operating characteristic analysis. RESULTS: Relative CBV best differentiated meningioma from glioblastoma and from metastasis with areas under the curve of 0.84 and 0.82, respectively. The percentage signal recovery best differentiated primary central nervous system lymphoma from metastasis with an area under the curve of 0.81. Relative CBV and percentage signal recovery were similar in differentiating primary central nervous system lymphoma from glioblastoma and from meningioma. Although neither relative CBV nor percentage signal recovery differentiated glioblastoma from metastasis, mean normalized baseline signal intensity achieved 86% sensitivity and 50% specificity. CONCLUSIONS: Similar to results for non-preload-based DSC-MR imaging, percentage signal recovery for one-quarter-dose preload-based, intermediate flip angle DSC-MR imaging differentiates most pair-wise comparisons of glioblastoma, metastasis, primary central nervous system lymphoma, and meningioma, except for glioblastoma versus metastasis. Differences in normalized post-preload baseline signal for glioblastoma and metastasis, reflecting a snapshot of dynamic contrast enhancement, may motivate the use of single-dose multiecho protocols permitting simultaneous quantification of DSC-MR imaging and dynamic contrast-enhanced MR imaging parameters.

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BACKGROUND AND PURPOSE: The accuracy of DSC-MR imaging CBV maps in glioblastoma depends on acquisition and analysis protocols. Multisite protocol heterogeneity has challenged standardization initiatives due to the difficulties of in vivo validation. This study sought to compare the accuracy of routinely used protocols using a digital reference object. MATERIALS AND METHODS: The digital reference object consisted of approximately 10,000 simulated voxels recapitulating typical signal heterogeneity encountered in vivo. The influence of acquisition and postprocessing methods on CBV reliability was evaluated across 6912 parameter combinations, including contrast agent dosing schemes, pulse sequence parameters, field strengths, and postprocessing methods. Accuracy and precision were assessed using the concordance correlation coefficient and coefficient of variation. RESULTS: Across all parameter space, the optimal protocol included full-dose contrast agent preload and bolus, intermediate (60°) flip angle, 30-ms TE, and postprocessing with a leakage-correction algorithm (concordance correlation coefficient = 0.97, coefficient of variation = 6.6%). Protocols with no preload or fractional dose preload and bolus using these acquisition parameters were generally less robust. However, a protocol with no preload, full-dose bolus, and low (30°) flip angle performed very well (concordance correlation coefficient = 0.93, coefficient of variation = 8.7% at 1.5T and concordance correlation coefficient = 0.92, coefficient of variation = 8.2% at 3T). CONCLUSIONS: Schemes with full-dose preload and bolus maximize CBV accuracy and reduce variability, which could enable smaller sample sizes and more reliable detection of CBV changes in clinical trials. When a lower total contrast agent dose is desired, use of a low flip angle, no preload, and full-dose bolus protocol may provide an attractive alternative.

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BACKGROUND AND PURPOSE: The optimal TE must be calculated to minimize the variance in CBV measurements made with DSC MR imaging. Simulations can be used to determine the influence of the TE on CBV, but they may not adequately recapitulate the in vivo heterogeneity of precontrast T2*, contrast agent kinetics, and the biophysical basis of contrast agent-induced T2* changes. The purpose of this study was to combine quantitative multiecho DSC MRI T2* time curves with error analysis in order to compute the optimal TE for a traditional single-echo acquisition. MATERIALS AND METHODS: Eleven subjects with high-grade gliomas were scanned at 3T with a dual-echo DSC MR imaging sequence to quantify contrast agent-induced T2* changes in this retrospective study. Optimized TEs were calculated with propagation of error analysis for high-grade glial tumors, normal-appearing white matter, and arterial input function estimation. RESULTS: The optimal TE is a weighted average of the T2* values that occur as a contrast agent bolus transverses a voxel. The mean optimal TEs were 30.0 ± 7.4 ms for high-grade glial tumors, 36.3 ± 4.6 ms for normal-appearing white matter, and 11.8 ± 1.4 ms for arterial input function estimation (repeated-measures ANOVA, P < .001). CONCLUSIONS: Greater heterogeneity was observed in the optimal TE values for high-grade gliomas, and mean values of all 3 ROIs were statistically significant. The optimal TE for the arterial input function estimation is much shorter; this finding implies that quantitative DSC MR imaging acquisitions would benefit from multiecho acquisitions. In the case of a single-echo acquisition, the optimal TE prescribed should be 30-35 ms (without a preload) and 20-30 ms (with a standard full-dose preload).

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A number of cytochrome P450 (P450) 2E1 substrates are known to show kinetic deuterium isotope effects of approximately 5 on Km (DK = DKm/HKm), but not on kcat, in rat liver microsomes (e.g. N-nitrosodimethylamine, ethanol, and CH2Cl2). We observed DKm values of 3-5 for recombinant human P450 2E1-catalyzed ethanol oxidation. Replacing NADPH and O2 with the oxygen surrogate cumene hydroperoxide yielded similar results. Ferric P450 2E1 reduction was fast (k >1000 min-1) even in the absence of substrate. These results indicate that the basis for the increase in Km is in the latter portion of the catalytic cycle. The intrinsic isotope effect (Dk) for ethanol oxidation was determined (competitively) to be 3.8, indicating that C-H bond cleavage is isotopically sensitive. Pre-steady-state studies showed a burst of product formation (k = 410 min-1), with the burst amplitude corresponding to the P450 concentration. Deuteration of ethanol resulted in an isotope effect of 3.2 on the rate of the burst. We conclude that product release is rate-limiting in the oxidation of ethanol to acetaldehyde by P450 2E1. The steady-state kinetics can be described by a paradigm in which the kcat approximates the rate of product release, and Km is an expression in which the denominator is dominated by the rate of C-H bond breaking.

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Aryl acetylenes have been investigated as inhibitors of cytochrome P450 (P450)-dependent alkoxyresorufin dealkylation dealkylation activities in liver microsomes prepared from rats exposed to beta-naphthoflavone, isosafrole, or phenobarbital. Many of the acetylenes investigated produce pseudo-first-order time-dependent and NADPH-dependent losses of the dealkylation activities characteristic of mechanism-based irreversible inactivation (suicide inhibition). Replacing the terminal hydrogen of aryl acetylenes with a methyl group to convert ethynes into propynes enhances the inhibition of P450 1A enzymes; in some instances, this modification converts a reversible inhibitor of P450s into a suicide inhibitor. In contrast, ethynes are more effective suicide inhibitors of P450 2B-dependent dealkylations than the corresponding propynes. Aryl acetylenes with an ethynyl group on the 2 position of naphthalene or on the 9 position of phenanthrene and arylalkyl acetylenes with alkyl chains containing 2, 3, or 4 methylene groups are selective inhibitors of P450 2B1/2B2 in liver microsomes from rats. Aryl acetylenes also act as suicide inhibitors of P450 1A2 in human liver microsomes, of purified P450 1A2 from rabbit or rat liver in reconstituted systems, and of purified recombinant human P450 1A2 and 1A1 in reconstituted systems. 4-(1-Propynyl)biphenyl (4PBi) inactivated P450 1A2-dependent ethoxyresourfin deethylation (EROD) activity in human liver microsomes in an NADPH-dependent process (k(inactivation), 0.23 min-1; KI, 2.3 microM). 4PBi also inactivated purified recombinant human P450 1A2 (k(inactivation), 0.24 min-1; KI, 4.3 microM). In agreement with previous reports [Yun, C.-H., Hammons, G. J., Jones, G., Martin, M. V., Hopkins, N. E., Alworth, W. L., and Guengerich, F. P. (1992) Biochemistry 31, 10556-10563], 2-ethynylnaphthalene (2EN) was not a suicide inhibitor of the P450 1A2 activity in human liver microsomes but did inactivate purified human P450 1A2. Neither 4PBi nor 2EN affected diagnostic activities of human microsomal P450 2E1, 2C9/10, 3A4, or 2C19. In the systems examined, the losses of P450-dependent activity produced by these aryl acetylenes were not accompanied by corresponding decreases in the measured P450 absorption spectra. Thus P450 inactivation by these aryl acetylenes does not involve labeling and destruction of the heme. Incubation of 4PBi with microsomal P450 1A1 or 1A2 from rat liver under conditions that lead to P450-dependent, enzyme inactivations generates a 2-biphenylylpropionic acid product. This suggests that the suicide inhibition of P450s by propynylaryl acetylenes proceeds via a methylaryl ketene formed by a 1,2-methyl rearrangement, analogous to the mechanism of suicide inhibition by ethynyl acetylenes that proceed via ketene intermediates formed by 1,2-hydrogen shifts [Ortiz de Montellano, P. R., and Kunze, K. L. (1981) Arch. Biochem. Biophys. 209, 710-712].

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An N-terminal block to Edman degradation was observed when any of five different mammalian cytochrome P450 (P450) proteins was expressed in Escherichia coli using the N-terminal sequence MALLLAVFL... This block was also seen in Salmonella typhimurium. With all proteins examined, the block could be removed by mild acid hydrolysis (0.6--6 N HCl, 23 degrees C) to expose Met as the N-terminus, suggesting N-formylMet retention. The N-terminal peptide of a modified P450 1A2 ("mutant 1", containing a thrombin-sensitive site inserted at residue 25) was released with thrombin and analyzed by electrospray mass spectrometry and found to yield the M(r) expected for the N-formyl derivative (+/- 0.8 amu). The region of positions 3--5 was altered by random mutagenesis, and three P450 1A2-expressing clones were analyzed for nucleotide and amino acid sequences. The changes from LLL were to RER (P450 1A2a), VDS (P450 1A2b), and WRH (P450 1A2c); these all show slightly dissimilar hydropathy plots compared to the MALLLAVFL... sequence. Mutant P450 1A2a had the N-terminal Met removed to yield N-terminal Ala; P450 1A2b contained an unmodified Met at the N-terminus; P450 1A2c had an approximately 80% block of the N-terminal Met. Experiments with bacterial membranes containing expressed P450 1A2 mutant 1 and P450 1A2 mutant 2 (thrombin-sensitive site inserted at residue 46) suggest that thrombin site 2, but not 1, is sequestered in the membrane. Spheroplasts of bacteria expressing P450 1A2 and the mutants at positions 3--5 were treated with proteinase K; amino acid analysis indicated that no cleavage occurred. These results are interpreted in a model in which most of the mammalian P450 expressed in the bacterium is located in the cytosol, the region near residue 46 is in the inner membrane, the region near residue 25 is in the cytosol, and the N-terminus is either imbedded in the membrane or free in the cytosolic space, depending upon the sequence. However, the possibility that the differences in N-terminal processing are the result of direct changes in interactions with the deformylase and Met aminopeptidase cannot be excluded.

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NADH-dependent 7-ethoxycoumarin O-deethylation activities could be reconstituted in systems containing cytochrome b5 (b5), NADH-b5 reductase, and bacterial recombinant P450 2E1 in 100 mM potassium phosphate buffer (pH 7.4) containing a synthetic phospholipid mixture and cholate. Replacement of NADH-b5 reductase with NADPH-P450 reductase yielded a 4-fold increase in 7-ethoxycoumarin O-deethylation activity, and further stimulation (approximately 1.5-fold) could be obtained when NADPH was used as an electron donor. Removal of b5 from the NADH- and NADPH-supported systems caused a 90% loss of 7-ethoxycoumarin O-deethylation activities in the presence of NADPH-P450 reductase, but resulted in complete loss of the activities in the absence of NADPH-P450 reductase. Km values were increased and Vmax values were decreased for 7-ethoxycoumarin O-deethylation when b5 was omitted from the NADPH-supported P450 2E1-reconstituted systems. Requirements for b5 in P450 2E1 systems were also observed in chlorzoxazone 6-hydroxylation, aniline p-hydroxylation, and N-nitrosodimethylamine N-demethylation. In human liver microsomes, NADH-dependent 7-ethoxycoumarin O-deethylation, chlorzoxazone 6-hydroxylation, aniline p-hydroxylation, and N-nitrosodimethylamine N-demethylation activities were found to be about 55, 41, 33, and 50%, respectively, of those catalyzed by NADPH-supported systems. Anti-rat NADPH-P450 reductase immunoglobulin G inhibited 7-ethoxycoumarin O-deethylation activity catalyzed by human liver microsomes more strongly in NADPH- than NADH-supported reactions, while anti-human b5 immunoglobulin G inhibited microsomal activities in both NADH- and NADPH-supported systems to similar extents. These results suggest that b5 is an essential component in P450 2E1-catalyzed oxidations of several substrates used, that about 10% of the activities occur via P450 2E1 reduction by NADPH-P450 reductase in the absence of b5, and that the NADH-supported system contributes, in part, to some reactions catalyzed by P450 2E1 in human liver microsomes.

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Rabbit cytochrome P450 1A2 was modified with succinimidyl carbonate poly(ethylene glycol) monomethyl ether, purified by size exclusion high performance liquid chromatography, and lyophilized. Modification of cytochrome P450 1A2 caused no structural deformation of the heme as evidenced by the similarity of the spectral signatures for both the ferric form and the ferrous-CO complex to the respective forms for the unmodified enzyme. Ethoxyresorufin O-deethylation activity in the presence of iodosobenzene for the modified enzyme was comparable to that of the native enzyme.

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The catalytic mechanism of cytochrome P450 (P450) enzymes has generally been understood in terms of a classic cycle in which electron donation is often limiting and catalysis is understood in terms of hydrogen abstraction and rapid oxygen rebound. In the course of detailed investigations with kinetic hydrogen isotope effects we have studied two systems in which somewhat unusual isotope effects have been interpreted in terms of modifications of the general paradigm. The low isotope effects observed for N-demethylation reactions are in contrast to high values seen with P450-catalyzed C-hydroxylation and peroxidase-catalyzed N-demethylation and are consonant with a role for the P450 FeO2+ entity in base-catalyzed deprotonation of an aminium radical. With P450 2E1, kinetic deuterium isotope effects are seen on the apparent Km for the substrate (increased) but not on Vmax. The results are interpreted in terms of a mechanism where C-H bond cleavage is sensitive to deuterium substitution but a step following this is rate-limiting. This step may be product release.

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The oxidation of benzo[a]pyrene (B[a]P) was examined using reconstituted systems prepared with recombinant human cytochrome P450 (P450) enzymes 1A1, 1A2, 2C8, 2C10, 2E1, and 3A4 and with microsomes prepared from Saccharomyces cerevisiae expressing recombinant human P450s 2C8, 2C9, and 2C18. Products measured by HPLC included the 3- and 9-phenols, the 4,5-, 7,8-, and 9,10-dihydrodiols (detected in the presence of epoxide hydrolase), and products in the polar fraction eluting immediately after the void volume. The most active enzyme in all reactions was P450 1A1. P450 3A4 and P450 1A2 formed appreciable amounts of several of the products, including the 3-phenol. P450 2C enzymes and P450 2E1 formed relatively low amounts of all B[a]P products. Consideration of these patterns along with knowledge of levels of expression of the P450s in human tissues and previous results with microsomes leads to the conclusion that P450 1A1 should dominate the oxidation of B[a]P in tissues where it is present and inducible. In human liver the level of P450 1A1 is low and P450 3A4, P450 2C subfamily enzymes, and P450 1A2 probably all contribute. Of the human P450s considered here, P450 1A2 was the most active hepatic enzyme forming the 7,8-dihydrodiol. 7,8-Benzoflavone stimulated the oxidation of B[a]P by P450 3A4 and inhibited the oxidations catalyzed by P450 1A2. The extent of inhibition of P450 1A1 was less (than with P450 1A2), probably due to the rapid oxidation of 7,8-benzoflavone by P450 1A1. The major 7,8-benzoflavone product appears to be the 5,6-oxide.

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Chlorate-resistant mutants of the denitrifying bacterium Thiosphaera pantotropha were generated by transposon Tn5 mutagenesis. One class was deficient in membrane-bound nitrate reductase activity but retained a periplasmic nitrate reductase activity. Using transposon marker rescue it was shown that in one such mutant, M-6, the transposon was inserted in the membrane-bound nitrate reductase beta subunit structural gene (termed narH in order to be consistent with the nomenclature of the Escherichia coli major nitrate reductase operon). The translated sequence (total of 106 amino acids) from around the point of transposon insertion showed approximately 90% amino acid identity with the beta subunits of the E. coli nitrate reductases. Under anaerobic growth conditions M-6 overproduced the periplasmic nitrate reductase activity allowing anaerobic growth with nitrate as electron acceptor. A regulatory link was inferred between the presence of the membrane-bound nitrate reductase and expression of the periplasmic nitrate reductase. This is the first demonstration of full denitrification in an organism possessing only a periplasmic nitrate reductase.

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Several strains of Rhodobacter capsulatus have been shown to possess a nitric oxide reductase activity (reaction product nitrous oxide) after anaerobic phototrophic growth, but not after aerobic growth. The reductase is associated with the cytoplasmic membrane and electrons can reach the enzyme via the cytochrome bc1 complex. However, use of appropriate strains has shown that neither the latter, cytochrome c2 nor cytochrome c' is essential for the reduction of nitric oxide. Inhibition by myxothiazol of nitric oxide reduction in a strain that lacks a cytochrome c2 establishes that in phototrophically grown R. capsulatus the cytochrome bc1 complex is able to transfer electrons to an acceptor that is alternative to cytochrome c2. Electron transport to nitric oxide from NADH or succinate generated a membrane potential. When isoascorbate plus 2,3,5,6-tetramethyl-p-phenylenediamine (DAD) was the electron donor a membrane potential was not generated. This observation implies that nitric oxide is reduced at the periplasmic surface of the membrane and that the reductase is not proton translocating.

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1. Addition of nitrous oxide to a periplasmic fraction released from Rhodobacter capsulatus strains MT1131, N22DNAR+ or AD2 caused oxidation of c-type cytochrome, as judged by the decrease in absorbance at 550 nm. The periplasmic fraction catalysed reduction of nitrous oxide in the presence of either isoascorbate plus phenazine ethosulphate or reduced methyl viologen. The rates with these two electron donors were similar and were comparable to the activity observed with a quantity of cells equivalent to those from which the periplasm sample had been derived. Activity in the periplasm could not be observed with ascorbate plus 2,3,5,6-tetramethyl-p-phenylenediamine although this reductant was effective with intact cells treated with myxothiazol to block the activity of the cytochrome-bc1 complex. 2. Cells of R. capsulatus MTG4/S4, a mutant from which the gene for cytochrome c2 has been specifically deleted, did not catalyse detectable rates of nitrous-oxide reduction. A nitrous-oxide reductase activity was present, as shown by activity of both cells and a periplasmic fraction with isoascorbate plus phenazine ethosulphate as reductant. The rates in cells and the periplasmic fraction were similar to those observed in the corresponding wild-type strain (MT1131). In contrast to wild-type cells, 2,3,5,6-tetramethyl-p-phenylenediamine and N,N,N',N'-tetramethyl-p-phenylenediamine [Ph(NMe2)2] were ineffective as mediators of electrons from isoascorbate. Visible absorption spectra showed that no detectable cytochromes in either the periplasm or intact cells of the MTG4/S4 mutant were oxidised by nitrous oxide. 3. Purified ferroycytochrome c2 from R. capsulatus was oxidised by nitrous oxide in the presence of periplasm from R. capsulatus MTG4/S4. The rate of oxidation was proportional to the amount of periplasm added, but was considerably lower than the rate of nitrous-oxide reduction observed with the same periplasmic fraction when either ascorbate plus phenazine ethosulphate or reduced methyl viologen were used as substrates. The oxidation of cytochrome c2 was inhibited by acetylene and by low concentrations of NaCl. 4. Oxidation of ferrocytochrome c2 by nitrous oxide was observed when the purified cytochrome was mixed with a preparation of nitrous-oxide reductase. However, oxidation of ferrocytochrome c' by nitrous oxide was not observed in the presence of the reductase. The observations with the mutant MTG4/S4 suggest that cytochrome c2 is the only periplasmic cytochrome involved in nitrous-oxide reduction. 5. Nitrous-oxide-dependent oxidation of a c-type cytochrome was observed in a periplasmic fraction from Paracoccus denitrificans, provided the fraction was first reduced.(ABSTRACT TRUNCATED AT 400 WORDS)

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Use of Clark-type electrodes has shown that, in cells of Thiosphaera pantotropha, the nitrous oxide reductase is active in the presence of O2, and that the two gases involved (N2O, O2) are reduced simultaneously, but with mutual inhibition. Reduction of nitrate, or nitrite, to N2O under aerobic conditions involves NO as an intermediate, as judged by trapping experiments with the ferric form of extracellular horse heart cytochrome c and the demonstration that the cells possess a nitric oxide reductase activity. The overall conversion of nitrate to N2, the process of denitrification, under aerobic conditions, is thus not prevented by reaction of NO with O2 and depends upon a nitrous oxide reductase system which differs from that in other organisms by being neither directly inhibited nor inactivated by O2.

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The unusual ability of Thiosphaera pantotropha to catalyze respiratory nitrate reduction under aerobic conditions is shown to correlate with the activity of a periplasmic nitrate reductase that is expressed under both aerobic and anaerobic growth conditions. The organism also synthesizes, but only under anaerobic conditions, a membrane-bound nitrate reductase which resembles the corresponding enzyme in Paracoccus denitrificans in respect of both catalytic properties and inhibition of activity in intact cells in the presence of oxygen.

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