Biological denitration of propylene glycol dinitrate by Bacillus sp. ATCC 51912.

In previous studies, bacterial cultures were isolated that had the ability to degrade the nitrate ester glyceryl trinitrate (i.e., nitroglycerin). The goal of the present study was to examine the ability of resting cells and cell-free extracts of the isolate Bacillus sp. ATCC 51912 to degrade the more recalcitrant nitrate ester propylene glycol dinitrate (PGDN). It was observed that the PGDN-denitrating activity was expressed during growth even when cells were cultured in the absence of nitrate esters. This indicates that nitrate esters are not required for expression of denitration activity. Using cell-free extracts, PGDN was observed to be sequentially denitrated to propylene glycol mononitrate (PGMN) and propylene glycol with the second denitration step proceeding more slowly than the first. Also it was observed that dialysis of the cell-free extracts did not affect denitration activity indicating that regenerable cofactors [e.g., NAD(P)H or ATP] are not required for denitration.

Denitration of glycerol trinitrate by resting cells and cell extracts of Bacillus thuringiensis/cereus and Enterobacter agglomerans.

A number of microorganisms were selected from soil and sediment samples which were known to have been previously exposed to nitrate ester contaminants. The two most effective bacteria for transforming glycerol trinitrate (GTN) were identified as Bacillus thuringiensis/cereus and Enterobacter agglomerans. For both isolates, denitration activities were expressed constitutively and GTN was not required for induction. Dialysis of cell extracts from both isolates did not affect denitration, which indicates that dissociable and depletable cofactors are not required for denitration. With thin-layer chromatography and high-performance liquid chromatography, the denitration pathway for both isolates was shown to be a sequential denitration of GTN to glycerol dinitrate isomers, glycerol mononitrate isomers, and ultimately to glycerol. GTN was observed to be completely converted to glycerol during a long-term incubation of cell extracts.

Isotopically sensitive regioselectivity in the oxidative deamination of a homologous series of diamines catalyzed by diamine oxidase.

The equivalence of aminomethylene groups in selected diamine substrates of diamine oxidase was exploited for the determination of intramolecular isotope effects. In the series of substrates, [1,1-2H2]-1,3-diaminopropane, [1,1-2H2]-1,5-diaminopentane, [1,1-2H2]-1,6-diaminohexane, [1,1-2H2]-1,7-diaminoheptane and [alpha,alpha-2H2]-4-(aminomethyl)benzylamine, the preference of the enzyme for reaction at the unlabeled methylene was found to vary from 1.45 to 10.5-fold. The observed partitioning ratios go through a minimum value with 1,5-diaminopentane, the best substrate of diamine oxidase of the compounds tested. The results suggest that fast substrates have less opportunity to reorient into alternate binding conformations while bound to the active site of the enzyme. On the other hand, diamine substrates tested that cannot exist in energetically favorable conformations with internitrogen distances of about 7-8 A showed larger intramolecular isotope effects.

A cyclic imine intermediate in the in vitro metabolic conversion of 1,6-diaminohexane to 6-aminohexanoic acid and caprolactam.

1. 3,4,5,6-Tetrahydro-2H-azepine is an intermediate in the enzyme-catalyzed conversion of 1,6-diaminohexane to 6-aminohexanoic acid and its corresponding lactam, caprolactam, by mammalian liver aldehyde oxidase. 2. Identification of metabolites was based on analysis by gas chromatography-mass spectrometry and confirmed by comparison with the properties of authentic standards. 3. The results indicate that the cell differentiating agent hexamethylene bisacetamide is converted into 1,6-diaminohexane, and its metabolism therefore involves diamine oxidase. 4. The metabolic fate of 1,6-diaminohexane is similar to that of putrescine and cadaverine in that a cyclic imine is an intermediate in the formation of metabolites with ring (lactam) and chain (amino acid) structures.

Induction of differentiation of human promyelocytic leukemia cells (HL60) by metabolites of hexamethylene bisacetamide.

We studied the ability of five metabolites of hexamethylene bisacetamide (HMBA), which we had previously identified in patient urine, to induce differentiation or to influence differentiation induced by HMBA of a human promyelocytic cell line. Differentiation of HL60 cells was quantified by morphological changes and by the ability to reduce nitroblue tetrazolium. N-Acetyl-1,6-diaminohexane (NADAH), the deacetylated, first metabolite of HMBA, was a more potent inducer of HL60 differentiation than was HMBA. NADAH produced 20-30% differentiation at 0.25 mM and 30-40% differentiation at 0.5 mM. NADAH (1 mM) induced 2-3-fold more differentiation than did 1 mM HMBA. HL60 differentiation, induced by various combinations of HMBA and NADAH, reflected a combined effect of the two compounds. In contrast, 1,6-diaminohexane, at 0.5-5 mM, failed to induce HL60 differentiation. Similarly, 0.5-5 mM 6-acetamidohexanoic acid, the major metabolite of HMBA, and 6-aminohexanoic acid failed to induce differentiation of HL60 cells. However, 6-acetamidohexanoic acid, when combined with HMBA or NADAH at various concentrations and ratios, enhanced the differentiation of HL60 cells induced by these two compounds. This enhancement was most apparent with addition of 0.50-3.0 mM 6-acetamidohexanoic acid to HL60 cells incubated with 1.0-3.0 mM HMBA or 0.25-1.0 mM NADAH. 6-Aminohexanoic acid similarly enhanced HMBA-induced differentiation of HL60 cells. These in vitro results have implications in terms of the clinical application of HMBA and interpretation of the results of clinical trials performed to date and may provide some insight into the mechanism of HMBA-induced cellular differentiation.

Involvement of monoamine oxidase and diamine oxidase in the metabolism of the cell differentiating agent hexamethylene bisacetamide (HMBA).

We have previously demonstrated a number of metabolites of hexamethylene bisacetamide (HMBA) in the urine of patients treated with HMBA. These include N-acetyl-1,6-diaminohexane (NADAH), 6-acetamidohexanoic acid (6AcHA), 1,6-diaminohexane (DAH) and 6-aminohexanoic acid (6AmHA). Because these compounds have potential roles in the dose-limiting metabolic acidosis and neurotoxicity associated with HMBA therapy, and are similar in structure to known substrates of monoamine oxidase (MAO) and diamine oxidase (DAO), we investigated the activities of these enzymes in the metabolic interconversion of HMBA metabolites. NADAH (5 mM) was incubated with MAO and aldehyde dehydrogenase. 6AcHA production was verified by gas chromatography-mass spectrometry and quantified by gas chromatography. 6AcHA production was linear for up to 4 hr. Complete inhibition of MAO activity was observed with 2 mM tranyl-cypromine or pargyline. Mouse liver microsomes, which do not contain MAO, did not convert NADAH to 6AcHA and, in control experiments, did not degrade 6AcHA. The HMBA metabolite, DAH, was a substrate for DAO, producing 3,4,5,6-tetrahydro-2H-azepine. Participation of DAO in the metabolism of HMBA implies potential interaction of HMBA and metabolites with polyamine metabolism and may represent a mechanism for HMBA's effects on cellular growth and differentiation. Metabolism of NADAH, also a differentiator, by MAO implies that concurrent use of HMBA and an MAO inhibitor may be clinically useful.

Plasma pharmacokinetics and urinary excretion of hexamethylene bisacetamide metabolites.

In order to further understand the clinical toxicities of hexamethylene bisacetamide (HMBA) and to allow appropriate in vitro studies, we developed a suitable gas chromatographic assay and quantified plasma concentrations and urinary excretion of four metabolites which we had previously identified in urine of patients receiving 5-day HMBA infusions at 4.8-43.2 g/m2/day. 6-Acetamidohexanoic acid (AcHA) was the major plasma metabolite and reached steady state concentration (Css) by 24 h. AcHA Css increased from 0.12 +/- 0.02 (SD) mM at 4.8 g/m2/day to 0.72 mM at 43.2 g/m2/day. The Css AcHA:Css HMBA ratio decreased with increasing HMBA dosage. At dosages below 24 g/m2/day plasma Css of N-acetyl-1,6-diaminohexane (NADAH), the initial metabolite of HMBA, were below the limit of detection of our assay. With HMBA infusions of 24, 33.6, and 43.2 g/m2/day, Css of NADAH were 0.16 +/- 0.05, 0.14 +/- 0.06, and 0.19 +/- 0.04 mM, respectively. Css NADAH:Css HMBA ratios at 24, 33.6, and 43.2 g/m2/day were 0.18 +/- 0.06, 0.08 +/- 0.02, and 0.31 +/- 0.05, respectively. Plasma Css of 1,6-diaminohexane and 6-aminohexanoic acid were below the limit of detection of our assay. Each patient's urinary excretion of NADAH, AcHA, and 1,6-diaminohexane was consistent from day to day. The fraction of dose excreted in urine as AcHA was not affected by HMBA dosage and accounted for 12.7 +/- 3.9% of the daily dose. The percentage of daily HMBA dose accounted for by excretion of NADAH decreased with increasing HMBA dosage (10.8 +/- 6.0% at 4.8 g/m2/day to 4.2 +/- 1.2% at 33.6 g/m2/day). Urinary excretion of 1,6-diaminohexane always accounted for less than 3% of the daily dose. Our results indicate that: (a) plasma concentrations of AcHA alone cannot explain the degree of acidosis observed with toxic doses of HMBA; (b) NADAH is present in plasma at concentrations that we have found to cause differentiation in vitro; and (c) the probable rate-limiting step in HMBA metabolism is the initial deacetylation.

Identification of metabolites of the cell-differentiating agent hexamethylene bisacetamide in humans.

Hexamethylene bisacetamide, a compound which in vitro induces differentiation in a wide variety of human and animal cancer cell lines, is being investigated in phase I clinical trials. After i.v. administration of hexamethylene bisacetamide to humans, urine contained the parent compound and at least five metabolites formed by deacetylation and oxidation pathways. Identification of urinary metabolites was accomplished by gas chromatography-mass spectrometric analysis after isolation by ion exchange chromatography or extraction with ethyl acetate. Metabolites with amino or alcohol groups were trifluoroacetylated and acidic functional groups were esterified with 2,2,2-trifluoroethanol or methanol. The structure of each metabolite was confirmed by comparison with authentic standards. Metabolites identified included the major metabolite, 6-acetamidohexanoic acid; the monodeacetylated product, N-acetyl-1,6-diaminohexane; the bis-deacetylated diamine, 1,6-diaminohexane; and the amino acid, 6-aminohexanoic acid and its lactam, caprolactam.

1-Piperideine as an in vivo precursor of the gamma-aminobutyric acid homologue 5-aminopentanoic acid.

Intraperitoneal injection of the cyclic imine 1-piperideine in mice resulted in measurable quantities of 5-aminopentanoic acid in brain. 5-Aminopentanoic acid is a methylene homologue of gamma-aminobutyric acid (GABA) that is a weak GABA agonist. 5-Aminopentanoic acid formed in the periphery was ruled out as the source of brain 5-aminopentanoic acid based on the absence of detection in brain following injection of 100 mg/kg of 5-aminopentanoic acid. Deuterium-labeled 1-piperideine was prepared by exchange in deuterated phosphate buffer. Injection of [3.3-2H2]1-piperideine yielded [2.2-2H2]5-aminopentanoic acid in brain. The results are consistent with uptake of 1-piperideine into brain and oxidation of the precursor to 5-aminopentanoic acid. Inhibition of GABA catabolism by pretreatment with aminooxyacetic acid increased brain concentrations of 5-aminopentanoic acid formed from 1-piperideine, suggesting that 5-aminopentanoic acid is an in vivo substrate of 4-aminobutyrate:2-oxoglutarate aminotransferase.

Biosynthesis of 5-aminopentanoic acid and 2-piperidone from cadaverine and 1-piperideine in mouse.

1-Piperideine, 5-aminopentanoic acid, and its lactam, 2-piperidone, were identified as metabolites of cadaverine in 10,000 g mouse liver supernatants to which diamine oxidase had been added. Both metabolites were also found when the cadaverine metabolite 1-piperideine was incubated with the preparation which suggested that 1-piperideine is an intermediate in the formation of 5-aminopentanoic acid and 2-piperidone. Identification of the metabolites was based on gas chromatography-mass spectrometric analysis in comparison to authentic standards. Mouse brain homogenates converted 1-piperideine to 5-aminopentanoic acid. The results suggest that the metabolic fate of cadaverine may provide precursors of pharmacologically active analogues of GABA.

Putrescine metabolism: enzymatic formation and non-enzymatic isotope exchange of delta1-pyrroline.

The deamination of putrescine catalysed by diamine oxidase was carried out in deuterium oxide and deuterated buffers. Enamine and alpha, beta-unsaturated intermediates were excluded, based on the observation that deuterium was not incorporated into delta 1-pyrroline during its enzymatic formation in deuterium oxide. When the reaction mixture was buffered with phosphate, isolated delta 1-pyrroline contained two deuterium atoms at C-3, indicating that a phosphate-promoted, non-enzymatic isotope exchange had occurred. Using 5,5-dimethyl-delta 1-pyrroline as a model compound, the nature of the non-enzymatic deuterium exchange was studied and a bifunctional catalysis mechanism proposed. The results suggest that the choice of buffer could alter the conclusions drawn from enzyme mechanism studies involving imine-enamine tautomerism .

Pyrrolines as prodrugs of gamma-aminobutyric acid analogues.

delta 1-Pyrroline, 5-methyl-delta 1-pyrroline, and 5,5-dimethyl-delta 1-pyrroline have been identified as substances metabolized to gamma-aminobutyric acid (GABA), 4-aminopentanoic acid (methylGABA), and 4-amino-4-methylpentanoic acid (dimethylGABA), respectively. An enzyme system residing in the soluble fraction of rabbit liver catalyzes the conversion of delta 1-pyrroline to GABA and its lactam, 2-pyrrolidinone. Acetaldehyde, allopurinol, and cyanide inhibited the reaction. Incubation of deuterium-labeled delta 1-pyrroline with mouse brain homogenates produced deuterated GABA. Mouse liver 10,000 g supernatant and mouse brain homogenates converted 5-methyl-delta 1-pyrroline to methylGABA, and 5,5-dimethyl-delta 1-pyrroline to dimethylGABA. Four hours after intraperitoneal injection of 5-methyl-delta 1-pyrroline (200 mg/kg), methylGABA was detected in mouse brain (0.27 mumol/g). DimethylGABA (1.21 mumol/g) was determined in mouse brain 30 min after intraperitoneal administration of 5,5-dimethyl-delta 1-pyrroline (200 mg/kg). Neither methylGABA nor dimethylGABA penetrated into the central nervous system when administered in the periphery. The present studies suggest that pyrrolines may represent a chemical class of brain-penetrating precursors of pharmacologically active analogues of GABA.

Applications of deuterium labeling in the study of the in vitro conversion of delta 1-pyrroline to 4-aminobutanoic acid and 2-pyrrolidinone.

delta 1-Pyrroline is a putrescine metabolite that is biotransformed by rabbit liver preparations to 4-aminobutanoic acid and its lactam, 2-pyrrolidinone. Analysis of dilute aqueous solutions of delta 1-pyrroline by proton nuclear magnetic resonance indicated the the predominating species in the liver incubation preparations was delta 1-pyrroline monomer, although other species, such as 4-aminobutyraldehyde an delta 1-pyrroline timer, may exist in equilibrium with the monomer. [2H12]-delta 1-Pyrroline trimer was synthesized from [2H5]pyrrolidine by conversion to the N-chloro derivative followed by dehydrohalogenation. 4-Aminobutanoic acid was measured by a gas chromatographic mass spectrometric assay after derivatization with dimethylformamide dimethyl acetal. The 4-aminobutanoic acid homologue, 5-aminovaleric acid, served as internal standard. 2-Pyrrolidinone was hydrolyzed and measured as 4-aminobutanoic acid. A comparison of the amounts of product formed following incubation of labeled and unlabeled delta 1-pyrroline indicated a significant isotope effect in the formation of 2-pyrrolidinone. The influence of the label was much less on 4-aminobutanoic acid production. The results suggest that there are two separate pathways involved in the reaction.

Detection of the in vivo conversion of 2-pyrrolidinone to gamma-aminobutyric acid in mouse brain.

Labeled gamma-aminobutyric acid was detected in mouse brain following intravenous injections of deuterium labeled 2-pyrrolidinone. [2H6]Pyrrolidinone was prepared by the reduction of [2H4]succinimide with lithium aluminum deuteride. Quantification was accomplished by a gas chromatography mass spectrometry assay method. gamma-Aminobutyric acid and internal standard, 5-aminovaleric acid, were converted to volatile derivatives by treatment with N,N-dimethylformamide dimethyl acetal. Quantitative estimates were derived from peak area measurements obtained from monitoring the parent ions of the gamma-aminobutyric acid and internal standard derivatives by repetitive scanning during the GC run. The conversion of pyrrolidinone to gamma-aminobutyric acid may provide a method for labeling central gamma-aminobutyric acid pools.