Methanol toxicity and formate oxidation in NEUT2 mice

NEUT2 mice are deficient in cytosolic 10-formyltetrahydrofolate dehydrogenase (FDH; EC 1.5.1.6) which catalyzes the oxidation of excess folate-linked one-carbon units in the form of 10-formyltetrahydrofolate to CO(2) and tetrahydrofolate (Champion et al., Proc. Natl. Acad. Sci. USA 91, 11338-11342, 1994). The absence of FDH should impair the oxidation of formate via the folate-dependent pathway and as a consequence render homozygous NEUT2 mice more susceptible to methanol toxicity. Normal (CB6-F1) and NEUT2 heterozygous and homozygous mice had essentially identical LD(50) values for methanol, 6.08, 6.00, and 6.03 g/kg, respectively. Normal mice oxidized low doses of [(14)C]sodium formate (ip 5 mg/kg) to (14)CO(2) at approximately twice the rate of homozygous NEUT2 mice, indicating the presence of another formate-oxidizing system in addition to FDH. Treatment of mice with the catalase inhibitor, 3-aminotriazole (1 g/kg ip) had no effect on the rate of formate oxidation, indicating that at low concentrations formate was not oxidized peroxidatively by catalase. High doses of [(14)C]sodium formate (ip 100 mg/kg) were oxidized to (14)CO(2) at identical rates in normal and NEUT2 homozygous mice. Pretreatment with 3-aminotriazole (1 g/kg ip) in this instance resulted in a 40 and 50% decrease in formate oxidation to CO(2) in both normal and homozygous NEUT2 mice, respectively. These results indicate that mice are able to oxidize formate to CO(2) by at least three different routes: (1) folate-dependent via FDH at low levels of formate; (2) peroxidation by catalase at high levels of formate; and (3) by an unknown route(s) which appears to function at both low and high levels of formate. The implications of these observations are discussed in terms of the current hypotheses concerning methanol and formate toxicity in rodents and primates.     

4-Aminobutyrate in mammalian putrescine catabolism

The effects of inhibitors of diamine oxidase (EC 1.4.3.6), monoamine oxidase (EC 1.4.3.4) and 4-aminobutyrate aminotransferase (EC 2.6.1.19) on the catabolism of putrescine in mice in vivo were studied. Diamine oxidase inhibitors and carboxymethoxylamine (amino-oxyacetate) markedly inhibit the metabolism of [(14)C]putrescine to (14)CO(2), but affect different enzymes. Aminoguanidine specifically inhibits the mitochondrial and non-mitochondrial diamine oxidases, whereas carboxymethoxylamine specifically inhibits 4-aminobutyrate transamination by the mitochondrial pathway. Hydrazine inhibits at both sites, and results in increased concentrations of 4-aminobutyrate in brain and liver. Pretreatment of mice with carboxymethoxylamine and [(14)C]putrescine leads to the urinary excretion of amino[(14)C]butyrate. Carboxymethoxylamine does not affect the non-mitochondrial pathway of putrescine catabolism, as the product of oxidative deamination of putrescine in the extramitochondrial compartment is not further oxidized but is excreted in the urine as derivatives of 4-aminobutyraldehyde. Another catabolic pathway of putrescine involves monoamine oxidase, and the monoamine oxidase inhibitor, pargyline, decreases the metabolism of [(14)C]putrescine to (14)CO(2)in vivo. Catabolism of putrescine to CO(2)in vivo occurs along different pathways, both of which have 4-aminobutyrate as a common intermediate, in contrast with the non-mitochondrial catabolism of putrescine, which terminates in the excretion of 4-aminobutyraldehyde derivatives. The significance of the different pathways is discussed.     

Regulation of histidine catabolism by succinate in Pseudomonas putida

The regulation of the histidine-degrading pathway is known to involve induction and repression. Our studies have shown that succinate may control the histidine-degrading pathway by sequential negative feedback inhibition. Succinate inhibited urocanase, and urocanate in turn inhibited histidase. Crude preparations of the two enzymes were made from Pseudomonas putida grown on l-histidine. Succinate was a competitive inhibitor of urocanase (K(i), 1.8 mm). Lactate, pyruvate, alpha-ketoglutarate, and glutamate did not inhibit urocanase. Urocanate inhibited histidase competitively (K(i), 0.13 mm). A multienzyme system (histidine to glutamate), when incubated with histidine and succinate, exhibited the combined effect. Succinate caused the level of accumulated urocanate to increase and indirectly blocked histidine disappearance. Growth of cells on urocanate as a nitrogen source was inhibited by 1% succinate. Succinate may play a physiological role in the biological regulation of histidine metabolism.     

Bioactivity of orally administered unnatural isomers, [6R]-5-formyltetrahydrofolate and [6S]-5,10-methenyltetrahydrofolate, in humans

It has been assumed that humans cannot utilize 5,6,7,8-tetrahydrofolates with the unnatural configuration at carbon 6, since these folates are enzymatically and microbiologically inactive. We hypothesized that orally administered unnatural [6R]-5-formyltetrahydrofolate or [6S]-5,10-methenyltetrahydrofolate is bioactive in humans. Subjects were given independent oral doses of these unnatural folates and of a natural [6S]-5-formyltetrahydrofolate. Plasma, before and after the dose for 4 h, and 2 h urine were collected. Areas under the curve for the change in plasma folate concentrations were measured microbiologically and urinary folates were measured using HPLC. Based on findings of plasma and urinary folates, the unnatural folates were estimated to be 14-50% active as compared to [6S]-5-formyltetrahydrofolate. The major plasma and urinary folate was [6S]-5-methyltetrahydrofolate in all experiments. In urine, a [6S]-5-formyltetrahydrofolate peak was observed only after a [6S]-5-HCO-H4folate dose and peaks of unnatural [6S]-10-formyltetrahydrofolate and 5-formyltetrahydrofolate were identified after a [6R]-5-formyltetrahydrofolate dose. A possible pathway that explains our findings is discussed. This pathway includes the oxidation of the unnatural [6S]-10-formyltetrahydrofolate to 10-formyl-7,8-dihydrofolate which can be further metabolized by 5-amino-4-imidazolecarboxamide-ribotide transformylase producing dihydrofolate. Dihydrofolate can then be metabolized to [6S]-5-methyltetrahydrofolate by well-established metabolism.     

Metabolism of sialic acids from exogenously administered sialyllactose and mucin in mouse and rat

A mixture of N-acetyl-[4,5,6,7,8,9-14C]neuraminosyl-alpha (2-3(6]-galactosyl-beta (1-4-glucose[( 14C]sialyl-lactose) and N-acetylneuraminosyl-alpha (2-3(6]-galactosyl-beta(1-4)-glucit-1-[3H]ol(sialyl-[3H]lactitol) as well as porcine submandibular gland mucin labeled with N-acetyl- and N-glycoloyl-[9-(3)H]neuraminic acid were administered orally to mice. The distribution of the different isotopes was followed in blood, tissues and excretion products of the animals. One half of the [14C]sialyl-lactose/sialyl-[3H]lactitol mixture given orally was excreted unchanged in the urine. The other half was hydrolysed by sialidase and partly metabolized further, followed by the excretion of 30% of the 14C-radioactivity as free N-acetyl-[4,5,6,7,8,9-14C]neuraminic acid and 60% of this radioactivity in the form of non-anionic compounds including expired 14CO2 within 24 h. The 14C-radioactivity derived from the [14C]sialyl-lactose/sialyl-[3H]lactitol mixture which remained in the bodies of fasted mice after 24 h was less than 1%. In the case of well-fed mice, a higher amount of the sialic acid residues was metabolized. The bulk of radioactivity of the mucin was resorbed within 24 h. About 40% of the radioactivity administered was excreted by the urine within 48 h; 30% of this radioactivity represented sialic acid and 70% other anionic and non-anionic metabolic products. 60% of the radioactivity administered remained in the body, and bound 3H-labeled sialic acids were isolated from liver. Sialyl-alpha (2-3)-[3H]lactitol was injected intravenously into rats; the substance was rapidly excreted in the urine without decomposition. These studies show that part of the sialic acids bound to oligosaccharides and glycoproteins can be hydrolysed in intestine by sialidase and be resorbed. This is followed either by excretion as free sialic acid or by metabolization at variable degrees, which apparently depends on the compound fed and on the retention time in the digestive tract.     

Substrate-mediated inactivation of urocanase from Pseudomonas putida. Evidence for an essential sulfhydryl group

Incubation of urocanase from Pseudomonas putida with either its substrate, urocanic acid, or product, 4'(5')-imidazolone-5'(4')-propionic acid, resulted in an oxygen-dependent inhibition of enzyme activity. Coincident with the inactivation was the stoichiometric incorporation of radioactivity from [14C]urocanate into the protein. NAD+ which is required for activity or urocanase was not directly involved in the inactivation process. The inactivation of urocanase was irreversible, could be partially blocked by the competitive inhibitor imidazolepropionate, and involved the modification of a single active-site thiol. The inhibition resulted from oxidative decomposition of 4'(5')-imidazolone-5'(4')-propionate but was not due to the formation of the major degradative product, 4-ketoglutaramate, since this compound was not an irreversible inactivator of urocanase although it did produce some inhibition at high concentrations. A mechanism is presented in which a reactive imine intermediate in the decomposition scheme is subject to nucleophilic attack by an active-site thiol, thereby generating a covalent enzyme--thioaminal adduct. These results emphasize the importance of a catalytic center sulfhydryl group for urocanase activity.     

Simultaneous determination of stable isotopically labelled l-histidine and urocanic acid in human plasma by stable isotope dilution mass spectrometry

A capillary gas chromatographic—mass spectrometric method for the simultaneous determination of stable isotopically labelled l-histidine (l-[3,3-²H₂,1′,3′-¹⁵N₂]histidine, l-His-[M + 4]) and urocanic acid ([3-²H,1′,3′-¹⁵N₂]urocanic acid, UA-[M + 3]) in human plasma was developed using dl-[2,3,3,5′-²H₄,2′-¹³C,1′,3′-¹⁵N₂]histidine (dl-His-[M + 7]) and [2,3,5′-²H₃,2′-¹³C,1′,3′-¹⁵N₂]urocanic acid (UA-[M + 6]) as internal standards. l-Histidine and urocanic acid were derivatized to ^αN-(trifluoroacetyl)-^imN-(ethoxycarbonyl)-l-histidine n-butyl ester and ^imN-(ethoxycarbonyl)urocanic acid n-butyl ester. Quantification was carried out by selected ion monitoring of the molecular ions of the respective derivatives of l-His-[M + 4], dl-His-[M + 7], UA-[M + 3] and UA-[M + 6]. The sensitivity, specificity, precision and accuracy of the method were demonstrated to be satisfactory for measuring plasma concentrations of l-His-[M + 4] and UA-[M + 3] following administration of trace amounts of l-His-[M + 4] to humans.     

The decarboxylation of S-adenosylmethionine by detergent-treated extracts of rat liver

1. The production of (14)CO(2) from S-adenosyl[carboxyl-(14)C]methionine by rat liver extracts was investigated. It was found that, in addition to the well-known cytosolic putrescine-activated S-adenosylmethionine decarboxylase, an activity carrying out the production of (14)CO(2) could be extracted from a latent, particulate or membrane-bound form by treatment with buffer containing 1% (v/v) Triton X-100 [confirming the report of Sturman (1976) Biochim. Biophys. Acta428, 56-69]. 2. The formation of (14)CO(2) by such detergent-solubilized extracts differed from that by cytosolic S-adenosylmethionine decarboxylase in a number of ways. The reaction by the solubilized extracts did not require putrescine and was not directly proportional to time of incubation or the amount of protein added. Instead, activity a showed a distinct lag period and was much greater when high concentrations of the extracts were used. The cytosolic S-adenosylmethionine decarboxylase was activated by putrescine, showed strict proportionality to protein added and the reaction proceeded at a constant rate. Cytosolic activity was not inhibited by homoserine or by S-adenosylhomocysteine, whereas the Triton-solubilized activity was strongly inhibited. 3. By using an acetone precipitate of Triton-treated homogenates as a source of the activity, it was found that decarboxylated S-adenosylmethionine was not present among the products of the reaction, although 5'-methylthioadenosine and 5-methylthioribose were found. Such extracts were able to produce (14)CO(2) when incubated with [U-(14)C]-homoserine, and (14)CO(2) production was greater when S-adenosyl[carboxyl-(14)C]methionine that had been degraded by heating at pH6 at 100 degrees C for 30min (a procedure known to produce mainly 5'-methylthioadenosine and homoserine lactone) was used as a substrate than when S-adenosyl[carboxyl-(14)C]methionine was used. 4. These results indicate that the Triton-solubilized activity is not a real S-adenosylmethionine decarboxylase, but that (14)CO(2) is produced via a series of reactions involving degradation of the S-adenosyl-[carboxyl-(14)C]methionine. It is probable that this degradation can occur via several pathways. Our results would suggest that part of the reaction occurs via the production of S-adenosylhomocysteine, which can then be converted into 2-oxobutyrate via the transsulphuration pathway, and that part occurs via the production of homoserine by an enzyme converting S-adenosylmethionine into 5'-methylthioadenosine and homoserine lactone.     

Reversible stepwise mechanism involving a carbanion intermediate in the elimination of ammonia from L-histidine catalyzed by histidine ammonia-lyase.

L-Histidine labeled with deuterium at the C-5' position of the imidazole ring, L-[5'-2H]histidine (His-5'-D), was used as a probe for investigating a stepwise reversible mechanism via a carbanion intermediate in the elimination of ammonia catalyzed by histidine ammonia-lyase (EC 4.3.1.3). The labeled L-histidine (His-5'-D) (2.45 mM) was incubated with histidine ammonia-lyase (200 units) from Pseudomonas fluorescens at pH 7.0 or 9.0 at 25.0 degrees C for 24 h. The time course of the reaction was examined to determine the rates of enzyme-catalyzed hydrogen exchange at C-5' of L-histidine and urocanic acid. The finding of the enzyme-catalyzed hydrogen exchange at C-5' of both L-histidine and urocanic acid in the presence of L-histidine provided a rational explanation for a stepwise reversible mechanism via a carbanion intermediate in the elimination reaction. The rate of increase in the concentration of urocanic acid exchanged with hydrogen (UA-5'-H) did not depend on the formation rate of urocanic acid and UA-5'-H was continuously formed at a constant rate (25.6 microM/h) even after the completion of urocanic acid formation. These observations suggested the presence of the reversible reaction of urocanic acid and a carbanion intermediate. Since there was only a minor contribution for the formation of UA-5'-H from L-histidine exchanged with solvent hydrogen (His-5'-H), the main pathway in the enzymatic reaction of His-5'-D must be the formation of UA-5'-D via a carbanion intermediate (carbanion-D). Regeneration of the carbanion-D from UA-5'-D by its reverse reaction and subsequent hydrogen incorporation at C-5' would contribute to a large extent for the formation of UA-5'-H. The stability of carbanion was also demonstrated to be approximately three times higher at pH 7.0 than at pH 9.0.     

Urocanic Acid Production by Sporogenous Bacteria

As the results of a screening of several type cultures of bacteria, Bacillus subtilis var. thermophilus was revealed to be the most powerful strain for urocanic acid production. The accumulation of urocanic acid by this bacteria is caused by deamination of L-histidine, and is particularly accelerated in the presence of a component (X-factor) in meat extract. In the decomposition of urocanic acid the optimal pH of urocanase activity is markedly inhibited by the deviation of pH of the culture medium. The histidase of this bacteria is supposed to be a new exo-type enzyme.     

Reassessment of 14CO2 compartmentation and of [14C]formate oxidation in rat liver

Our previous report (Marsolais, C., Huot, S., David, F., Garneau, M., and Brunengraber, H. (1987) J. Biol. Chem. 262, 2604-2607) had concluded that a fraction of [14C]formate oxidation in liver occurs in the mitochondrion. This conclusion was based on the labeling patterns of urea and acetoacetate labeled via 14CO2 generated from [14C]formate and other [14C]substrates. We reassessed our interpretation in experiments conducted in (i) perifused mitochondria and (ii) isolated livers perfused with buffer containing [14C]formate, [14C]gluconolactone, 14CO2, or NaH13CO3, in the absence and presence of acetazolamide, an inhibitor of carbonic anhydrase. Our data show that the cytosolic pools of bicarbonate and CO2 are not in isotopic equilibrium when 14CO2 is generated in the cytosol or is supplied as NaH14CO3. We retract our earlier suggestion of a mitochondrial site of [14C]formate oxidation.     

Cysteinesulfonate and beta-sulfopyruvate metabolism. Partitioning between decarboxylation, transamination, and reduction pathways

L-Cysteinesulfonate (L-cysteate) is present in plasma, urine, and tissues in concentrations comparable to that of L-cysteinesulfinate, the primary oxidative metabolite of L-cysteine. Although cysteinesulfonate is known to be decarboxylated to taurine by cysteinesulfinate decarboxylase, the occurrence and importance of other metabolisms has not been examined. The present studies indicate that cysteinesulfonate partitions in vivo between decarboxylation and transamination; the latter reaction is catalyzed by aspartate aminotransferase and yields beta-sulfopyruvate. Whereas beta-sulfinylpyruvate, the product of cysteinesulfinate transamination, decomposes spontaneously, beta-sulfopyruvate is stable and is reduced by malate dehydrogenase to beta-sulfolactate. When L-[1-14C]cysteinesulfonate is given to mice, 60-75% is decarboxylated to taurine and about 25% is excreted in the urine as beta-sulfolactate. beta-Sulfo[1-14C] pyruvate is found to partition about equally between beta-sulfolactate and cysteinesulfonate formation; greater than 90% of the latter is decarboxylated. Parenterally administered beta-sulfo[1-14C]lactate is mostly excreted in the urine, but 12% is metabolized via beta-sulfopyruvate and cysteinesulfonate to 14CO2 and taurine. beta-Sulfopyruvate is not excreted, and only traces of sulfoacetate, perhaps formed by oxidative decarboxylation, are detected. These studies establish that cysteinesulfonate, beta-sulfopyruvate, and beta-sulfolactate are reversibly interconverted in vivo. Since only cysteinesulfonate is directly metabolized to CO2, the rate of 14CO2 formation from L-[1-14C]cysteinesulfonate is a valid measure of total cysteinesulfinate decarboxylase activity in vivo; use of this assay permits inhibitor effects to be accurately determined in intact mice. Thus, whereas in vitro assays indicate that beta-methyleneaspartate inhibits brain, liver, and kidney cysteinesulfinate decarboxylase by 0, greater than 60, and 90%, respectively, in vivo studies with L-[1-14C]cysteinesulfonate show net metabolic inhibition is about 40%.     

The biological fate of vinylidene chloride in rats

The main eliminative route for [14C] vinylidene chloride ([14C]DCE) after intragastric, i.v. or i.p. administration to rats is pulmonary; both unchanged DCE and DCE-related CO2 are excreted by that route and other DCE metabolites via the kidneys. Part of the urinary 14C is of biliary origin. After intragastric dosing, the plot of the pulmonary output of unchanged DCE against the logarithm of reciprocal doses in biphasic. Pulmonary elimination of DCE and CO2 and urinary excretion of DCE metabolites after an intragastric dose occupy 3 days. In comparison, 80% of a small i.v. dose is excreted unchanged within 1 h of injection; more than 60% within 5 min. Biotransformation of DCE affords thiodiglycollic acid, and an N-acetyl-S-cysteinyl-acetyl derivative as major urinary metabolites together with substantial amounts of chloroacetic acid, dithioglycollic acid and thioglycolic acid. It is probable that chloroacetic acid, which is a DCE metabolite per se, lies on a main metabolic pathway for DCE, since it affords several metabolites in common with DCE. Furthermore, electrolysis of one molecular proportion of the [14C]thiodiglycollate metabolite from [1(-14)C]DCE or [1(-14C]chloroacetic acid gives 1 equivalent of 14CO2, and this evidence is consistent with the transformation of DCE into chloroacetic acid by a mechanism involving the migration of one Cl atom and the loss of the other one. CO2 (and hence urea) may be produced through the action of epoxide hydratase on 1,1-dichloroethylene oxide or by a minor oxidative pathway for chloroacetic acid. The N-acetyl-S-cysteinyl-acetyl derivative is probably formed via the reaction of 1,1-dichloroethylene oxide and glutathione S-epoxide transferase.     

Studies on the production and assessment of experimental histidinemia in the rat

Intraperitoneal administration to rats of D- or DL-α-hydrazunoimidazolylpropionic acid was found to produce a substantial inactivation of hepatic histidine ammonia-lysase (EC 4.3.1.3) in vivo. Proportional to this loss in enzyme activity was an impairment of the ability of treated rats to oxidize l-[ring-2-¹⁴C] histidine to ¹⁴CO₂. Rats in which hepatic histadine ammonia-lyase activity was either depressed by dl-hydrazunoimidazolylproprionic acid injection or elevated by feeding a high protein diet displayed proportionately altered rates of ³H₂O release into plasma water following l-[3-H]histidine administration. Plasma l-histidine clearance following loading with this amino acid was similarly affected by these treatments. Administration of dl-α-hydrazinoimisazolyl-proprionic acid to rats was also found to inactivate non-specifically pyridoxal 5-phosphate enzymes in vivo; pyridoxine injection was found to reverse the dl-α-hydrazinoimidazolylproprionic acid-induced inactivation of hepatic aspartate aminotransferase (EC 2.6.1.1) in vivo, but not that of hepatic histidine ammonia-lyase. These findings demonstrate that histidine ammonia-lyase is the rate-limiting factor in l-histidine degradation in the rat. The potential usefulness of dl-hydrazinoimidazolylproprionic acid in the production of an animal model for histidinemia (hereditary histidine ammonia-lyase deficiency) is discussed.     

N tau methyl histidine excretion and [U-14C]amino acid oxidation in fully chickens from two lines selected for high and low body fat contents

Protein turnover and amino acid oxidation were examined in two lines of domestic broiler chicken selected for high (fat) and low (lean) plasma very low density lipoprotein levels. Protein turnover was assessed by comparing N tau methyl histidine excretion in two lines. No differences between the fat and lean birds were found. Oxidation of [U-14C]amino acids in vivo was measured in birds of both lines which had been fed ad lib. The lean birds oxidized significantly less of the administered [U-14C]amino acids to 14CO2 than their fat counterparts during the first 90 min following administration. Lean birds also excreted significantly less 14C than fat birds from [U-14C]amino acids.     

Gluconate metabolism in germinated spores of Bacillus megaterium QM B1551: primary roles of gluconokinase and the pentose cycle

The metabolic pathway of gluconate, a major product of glucose metabolism during spore germination, was investigated in Bacillus megaterium QM B1551. Compared to the parent, mutant spores lacking gluconokinase could not metabolize gluconate, whereas the revertant simultaneously restored the enzyme activity and the ability to metabolize it, indicating that gluconokinase was solely responsible for the onset of gluconate metabolism. To identify a further metabolic route for gluconate, we determined 14C yields in acetate and CO2 formed from [14C]gluconate, and found that experimental ratios of 14CO2/[14C]acetate obtained from [2-14C]gluconate and [3,4-14C]gluconate were not compatible with the ratios predicted from the Entner-Doudoroff pathway. In contrast, when CO2 release caused by recycling (approx. 30%) was corrected, the ratios almost agreed with those from the pentose cycle. Comparison of specific radioactivities in acetate also supported the conclusion that gluconate was metabolized via the pentose cycle, subsequently metabolized via the Embden-Meyerhof pathway, and finally degraded to acetate and CO2 without a contribution by the Krebs cycle.     

Metabolic distribution of radioactivity in Sprague-Dawley rats and B6C3F1 mice exposed to 1,3-[2,3-14C]-butadiene by whole body exposure

The uptake of 1,3-[2,3-(14)C]-butadiene and its disposition, measured as radioactivity in urine, faeces, exhaled volatiles and CO(2) during and following 6 h whole body exposure to 20 ppm butadiene has been investigated in male Sprague-Dawley rats and B6C3F1 mice. Whilst there were similarities between the two species, the uptake and metabolic distribution of butadiene were somewhat different for rats and mice. The major differences observed were in the urinary excretion of radioactivity and in the exhalation of 14C-CO(2). After 42 h from the start of exposure, 51.1% of radioactivity was eliminated in rat urine compared with 39.5% for mouse urine. 34.9% of the recovered radioactivity was exhaled by rats as 14C-CO(2), compared with 48.7% by mice. Excretion of radioactivity in faeces was similar for both species (3.8% for rats and 3.4% for mice). The tissue concentrations of 14C-butadiene equivalents measured in liver, testes, lung and blood of exposed mice were 0.493, 0460, 0.457, and 1.626 nmol/g tissue, respectively. The values for the corresponding rat tissues were 0.869, 0.329, 0.457, and 1.626 nmol butadiene equivalents/g tissue, respectively. For rats, 6.2% of recovered radioactivity (0.288 nmol butadiene equivalents/g tissue) was retained in carcasses whereas for mice the amount was 3.6% (0.334 nmol butadiene equivalents/g tissue). There were also some significant differences between the metabolic conversion of 1,3-[2,3-(14)C]-butadiene and excretion by mice following the 20 ppm whole body exposure compared to previously reported data for nose-only exposure to 200 ppm butadiene [Richardson et al., Toxicol. Sci. 49 (1999) 186]. The main difference between the high- and low-exposure studies was in the exhalation of 14C-CO(2). At the 200 ppm exposure, 40% of the radioactivity was exhaled as 14C-CO(2) by rats whereas 6% was measured by this route for mice. The proportional conversion of butadiene to CO(2) by mice was significantly greater at the low exposure concentration compared with that reported for the higher concentration. This shift was not observed for rats. The difference between species could be caused by a saturation of metabolism in mice between 20 and 200 ppm for the pathways leading to CO(2). Restraint or error in collection of CO(2) in the 200 ppm study could also be factors.     

The control of the enzymes degrading histidine and related imidazolyl derivatives in Pseudomonas testosteroni

1. The induction of the enzymes for the degradation of l-histidine, imidazolylpropionate and imidazolyl-l-lactate in Pseudomonas testosteroni was investigated. 2. The activities of histidine ammonia-lyase, histidine-2-oxoglutarate aminotransferase and urocanase are consistent with these enzymes being subject to co-ordinate control under most growth conditions. However, a further regulatory mechanism may be superimposed for histidase alone under conditions where degradation of histidine must take place for growth to occur. 3. Experiments with a urocanase(-) mutant show that urocanate is an inducer for the enzymes given above and also for N-formiminoglutamate hydrolyase and N-formylglutamate hydrolase. 4. N-Formiminoglutamate hydrolase and N-formylglutamate hydrolase are also induced by their substrates, and it is suggested that these two enzymes may be different gene products from those expressed in the presence of urocanate. 5. Induction of the enzyme system for the oxidation of imidazolylpropionate is dependent on exposure of cells to this compound.     

Oxidation of glucose, ribose, alanine, and glutamate by Leishmania braziliensis panamensis

The metabolism of [1-14C]- and [6-14C]glucose, [1-14C]ribose, [1-14C]- and [U-14C]alanine, and [1-14C]- and [5-14C]glutamate by the promastigotes of Leishmania braziliensis panamensis was investigated in cells resuspended in Hanks' balanced salt solution supplemented with ribose, alanine, or glutamate. The ratio of 14CO2 produced from [1-14C]glucose to that from [6-14C]glucose ranged from about two to six, indicating appreciable carbon flow through the pentose phosphate pathway. A functional pentose phosphate pathway was further demonstrated by the production of 14CO2 from [1-14C]ribose although the rate of ribose oxidation was much lower than the rate of glucose oxidation. The rate of 14CO2 production from [1-14C]glucose was almost linear with time of incubation, whereas that of [6-14C]glucose accelerated, consistent with an increasing rate of flux through the Embden-Meyerhof pathway during incubation. Increasing the assay temperature from 26 degrees C to 34 degrees C had no appreciable effect on the rates or time courses of oxidation of either [1-14C]- or [6-14C]glucose or of [1-14C]ribose. Both alanine and glutamate were oxidized by L. b. panamensis, and at rates comparable to or appreciably greater than the rate of oxidation of glucose. The ratios of 14CO2 produced from [1-14C]- to [U-14C]alanine and from [1-14C]- to [5-14C]glutamate indicated that these compounds were metabolized via a functioning tricarboxylic acid cycle and that most of the label that entered the tricarboxylic acid cycle was oxidized to carbon dioxide.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effects of vitamin A deficiency on hepatic folate metabolism in rats

The effects of severe vitamin A deficiency (liver retinol less than 2 micrograms/g) on hepatic folate metabolism in rats were studied. The oxidation of a [ring-2-14C] histidine load or a [14C]formate load to 14CO2 was significantly depressed in vitamin A-deficient rats and those given histidine also excreted more urinary formiminoglutamic acid (FiGlu) than pair-fed controls. The increase in FiGlu excretion was not due to augmented production from histidine, implicating an impairment of FiGlu catabolism. FiGlu formiminotransferase activity was unaltered in vitamin A-deficient rats, but hepatic tetrahydrofolic acid (THF) concentration was decreased by 58% in vitamin A-deficient rats given a histidine load while 5-methyl-THF concentration was increased by 39%. Formyl-THF and total folate levels were similar to controls. A redistribution of folate coenzymes was not found in vitamin A-deficient rats not force fed histidine. A 43% decrease in 10-formyl-THF dehydrogenase activity, which generates both THF and the 14CO2 from the labeled substrates, and an 81% increase in 5,10-methylene-THF reductase activity, which generates 5-methyl-THF, were found in vitamin A-deficient rats. It appears that the production of severe vitamin A deficiency results in selective changes in the activities of hepatic folate-dependent enzymes, so that when a load of a one-carbon donor is given, THF concentration decreases and metabolism of the load is impaired.