Bile acid synthesis in man. In vivo activity of the 25-hydroxylation pathway.

During biosynthesis of bile acid, carbons 25-26-27 are removed from the cholesterol side-chain. Side-chain oxidation begins either with hydroxylation at the 26-position, in which case the three-carbon fragment is released as propionic acid, or with hydroxylation at the 25-position, in which case the three-carbon fragment is released as acetone. We have previously shown in the rat that the contribution of the 25-hydroxylation pathway can be quantitated in vivo by measuring production of [14C]acetone from [14C]26-cholesterol. In the present study, we adapted this method to human subjects. 4 d after oral administration of 100 microCi of [14C]26-cholesterol and 1 d after beginning a constant infusion of 16.6 mumol/min unlabeled acetone, three men and two women underwent breath collections. Expired acetone was trapped and purified as the 2,4 dinitrophenylhydrazine derivative. 14CO2 was trapped quantitatively using phenethylamine. Specific activity of breath acetone was multiplied by the acetone infusion rate to calculate production of [14C]acetone. [14C]Acetone production averaged 4.9% of total release of 14C from [14C]26-cholesterol, estimated by 14CO2 output. The method was validated by showing that [14C]acetone production from [14C]isopropanol averaged 86.9% of the [14C]-isopropanol infusion rate. We conclude that in man, as in the rat, the 25-hydroxylation pathway accounts for less than 5% of bile acid synthesis.

[Selection of mutants of Clostridium acetobutylicum defective in production of acetone]. / Sélection de mutants de Clostridium acetobutylicum défectifs dans la production d'acétone.

The butyrate analogue 2-bromobutyrate is toxic toward Clostridium acetobutylicum. After mutation using nitrosoguanidine, mutants resistant to the suicide co-substrate 2-bromobutyrate were selected. Such mutants no longer produced acetone, whereas the synthesis of butanol and ethanol was unaffected. Enzymatic analysis of a 2-bromobutyrate-resistant mutant showed a low level of coenzyme A transferase and of acetoacetate decarboxylase, the two enzymes implicated in the biosynthesis of acetone, whereas the level of hydrogenase was unaffected.

Effects of butyric and acetic acids on acetone-butanol formation by Clostridium acetobutylicum.

The actions of butyric and acetic acids on acetone-butanol fermentation are investigated. Production of butyric and acetic acids are controlled by the extracellular concentrations of both acids: acetic acid added to the medium inhibits its own formation but has no effect on butyric acid formation, and added butyric acid inhibits its own formation but not that of acetic acid. The ratio of end metabolites depends upon acetic and butyric acid quantities excreted during the fermentation. In contrast to acetic acid, which specifically increases acetone formation, butyric acid increases both acetone and butanol formations. Acetate and butyrate kinase activities were also examined. Both increase at the start of fermentation and decrease when solvents appear in the medium. Coenzyme A transferase activity is weak in the acidogenic phase and markedly increases in the solvent phase. Acetic and butyric acids appear to be co-substrates. On the basis of these results, a mechanism of acetic and butyric acid pathways, coupled to solvent formation by C. acetobutylicum glucose fermentation is proposed.

Rat acetoacetic acid decarboxylase inhibition by acetone.

Although in mammals, acetone formation from acetoacetic acid is normally regarded as a non-enzymatic (spontaneous) process, the existence of an acetoacetic acid decarboxylase activity was postulated recently. The results imply that this enzymatic activity can be relatively important at the physiological concentrations of ketone bodies found in the rat and that acetone acts as a competitive inhibitor of this enzyme.

[Acetobutylic fermentation: strains and regional raw materials]. / Fermentación acetobutílica: cepas y materias primas regionales.

The purpose of the present work was to show, as a first stage, that it is possible to characterize autochtohnous strains of Clostridium acetobutilicum of a good solvent producing capacity, specially N-butanol, through the utilization of suitable techniques for isolating anaerobic microorganisms. Cassava roots were employed as raw material using suitable culture media and an anaerobic jar of cold catalyst. The fermentative capacity of the strains thus isolated was evaluated against a control strain of Clostridium acetobutilicum. Even though some of the strains showed a greater solvent producing power, most of them showed lower fermentation capacity than the control strain, which could be increased, by applying successive thermic treatments. As a second stage, and due to the low cost production of cassava in the Province of Misiones, we studied its utilization as an acetone-butanol fermentation substrate. Mashes composed of binary mixtures of cassava flour and variable amounts of integral flour maize or soy were treated with selected "starters" of Clostridium acetobutilicum, being further processed according to standardized techniques in order to obtain the already mentioned solvents. Mashes concentration influence was also studied using culture media the composition of which proved to be excellent in all experiments carried out under "static system" conditions. The highest fermentative yields (maximum value recorded: 26,20 g of total solvents, with respect to dry solids), were recorded for mashes obtained from mixtures containing integral maize flour; these showed a higher degree of nutrients utilization than those prepared with integral soy flour.

Effect of pyruvate decarboxylase activity and of pyruvate concentration on the production of 1-hydroxy-1-phenylpropanone in Saccharomyces carlsbergensis.

The assumption that the pyruvate decarboxylase activity of Saccharomyces carlsbergensis is the main limiting factor determining the formation rate and the total amount of d(--)-l-hydroxy-l-phenyl-propanone (phenylacetylcarbinol, PAC) produced was not confirmed. An increase of about 30% of the total amount of the PAC produced was obtained when 8.5% sodium pyruvate was gradually added. The total PAC production is probably influenced both by the pyruvate decarboxylase activity and the pyruvate concentration in the cells, the latter being actually the determining rate-limiting factor.

Influence of cycloheximide on normal and induced delta-aminolevulinic acid synthetase (aminoketone synthesis) of rat liver in vivo and in vitro.

2 and 5 mg/kg cycloheximide given i.p. 30 minutes prior to the inducer allylisopropylacetamide (AIA) diminished or prevented the enhancement of delta-aminolevulinic acid synthetase (ALAS) activity in newborn rats. In adult rats the small AIA effect 24 hours after pretreatment was enhanced by 2 mg/kg cycloheximide i.p., when the inhibitor was injected 2 hours prior to the inducer. The basic aminoketone synthesis was inhibited by cycloheximide alone. 150-600 microgram/ml cycloheximide had no effect on the aminoketone synthesis of rat liver slices in vitro during 4 hours of incubation. The in vitro induction by 0.6 mg/ml AIA was enhanced by cycloheximide, if the inducer was added 1 hour after preincubation with cycloheximide. It is supposed, that the cytoplasmically localized precursor-ALAS is activated at the beginning of the induction process prior to the initiation of de novo enzyme synthesis, at least in vitro.

Glucose and fructose metabolism in Zymomonas anaerobia.

Isotopic and enzymic evidence indicates that Zymomonas anaerobia ferments glucose via the Entner-Doudoroff pathway. The molar growth yields with glucose (5.89) and fructose (5.0) are lower than those for the related organism Zymomonas mobilis and the observed linear growth suggests that energetically uncoupled growth occurs. A survey of enzymes of carbohydrate metabolism revealed the presence of weak phosphofructokinase and fructose 1,6-diphosphate aldolase activities but phosphoketolase, transketolase and transaldolase were not detected. Fermentation balances for glucose and fructose are reported; acetaldehyde accumulated in both fermentations, to a greater extent with fructose which also yielded glycerol and dihydroxyacetone as minor products.

Biphasic growth of Acetobacter suboxydans on a glycerol-limiting medium.

Dihydroxyacetone was quantitatively produced from glycerol during the primary exponential growth phase and depleted during the secondary exponential phase. Although no growth was detected on the basal medium, growth occurred upon addition of dihydroxyacetone.

The biology of methyl ketones.

Examples of the biological occurrence of methyl ketones are reviewed. The lack of significant accumulations of these compounds in the biosphere indicates that a recycling of these organic molecules is occurring. Evidence for biodegradation of acetone by mammals and longer methyl ketones by microorganisms via terminal methyl-group oxidation is discussed. A new mechanism for the subterminal oxidation of methyl ketones by microorganisms is proposed whereby the first intermediate produced is an acetate ester which subsequently is cleaved to acetate and a primary alcohol two carbons shorter than the original ketone substrate. Methyl ketones can be produced by mammals and fungi by decarboxylation of beta-keto acids. Some bacteria are able to form methyl ketones via the oxidation of aliphatic hydrocarbons at the methylene carbon alpha to the methyl group. Speculations on the biosynthesis of methyl ketones by insects and plants and a discussion of the possible biological roles of methyl ketones in diverse biological systems are presented.

The limitation of glycolysis in adenine-deficient Escherichia coli.

1. In many instances, there was no change in the rate of oxygen consumption per cell when adenine was withdrawn from purine auxotrophs of Escherichia coli and Salmonella typhimurium. 2. However, adenine deficiency inhibited the metabolism of glucose, mannitol or glycerol in a purA(-) strain, in purB(-) or purH(-) strains in the absence of histidine and in purB(-) mutants supplied with hypoxanthine. These are all instances where reactions occur to consume adenine nucleotides. 3. The inhibition of glucose oxidation is accompanied by the accumulation of fructose 1,6-diphosphate and dihydroxyacetone phosphate. 4. Insufficiency of ADP for phosphoglycerate kinase is the most probable cause of the inhibition.