14C-labeled substrate catabolism by human diploid fibroblasts derived from infants and adults

Untransformed diploid skin fibroblasts from eight normal adults, aged 24 to 74 years, catabolized several 14C-labeled substrates less effectively than cells from ten normal male infants. 14C-labeled substrate metabolism was quantitated either by measuring the evolution of 14CO2 from the 14C-labeled compounds or the incorporation of 14C into cellular protein via transamination of tricarboxylic acid cycle intermediates derived from the 14C-labeled substrates. With these methods, adult cells catabolized [1-14C]butyrate, [1-14C]octanoate, and 1-[2-14C]leucine at rates 44 to 64% of those found in infant cells. The oxidation of [1,4-14C]succinate and [U-14C]malate was identical in both infant and adult cells, while [2,3-14C]succinate catabolism was mildly decreased in adult cells (65-80% of control). These observations parallel those made in rat tissues and confirm that the same phenomenon occurs in cultured human fibroblasts.     

14CO2 production is no adequate measure of [14C]fatty acid oxidation

Palmitate oxidation was comparatively assayed in various cell-free and cellular systems by 14CO2 production and by the sum of 14CO2 and 14C-labeled acid-soluble products. The 14CO2 production rate was dependent on incubation time and amount of tissue in contrast to the total oxidation rate. The 14CO2 contribution to the oxidation rate of [1-14C]palmitate varied with homogenates from 1% with rat liver to 28% with rat kidney and amounted to only 2-4% with human muscles. With cellular systems the 14CO2 contribution varied between 20% in human fibroblasts and 70% in rat muscles and myocytes. Addition of cofactors increased the oxidation rate, but decreased the 14CO2 contribution. Various conditions appeared also to influence to a different extent the 14CO2 production and the total oxidation rate with rat tissue homogenates and with rat muscle mitochondria. Incorporation of radioactivity from [1-14C]palmitate into protein was not detectable in cell-free systems and only 2-3% of the sum of 14CO2 and 14C-labeled acid-soluble products in cellular systems. Assay of 14CO2 and 14C-labeled acid-soluble products is a much more accurate and sensitive estimation of fatty acid oxidation than assay of only 14CO2.     

14C-most-probable-number method for enumeration of active heterotrophic microorganisms in natural waters.

A most-probable-number method using 14C-labeled substrates is described for the enumeration of aquatic populations of heterotrophic microorganisms. Natural populations of microorganisms are inoculated into dilution replicates prepared from the natural water from which the organisms originated. The natural water is supplemented with a 14C-labeled compound added so as to approximate a true environmental concentration. 14CO2 evolved by individual replicates is trapped in NaOH and counted by liquid scintillation techniques for use in scoring replicates as positive or negative. Positives (14CO2 evolution) are easily distinguished from negatives (no 14CO2 evolution). The results from a variety of environments using the 14CO2 procedure agreed well with previously described methods, in most instances. The 14C-most-probable-number method described here reduces handling procedures over previously described most-probable-number procedures using 14C-labeled substrates. It also appears to have advantages over other enumeration methods in its attempt to approximate natural conditions more closely.     

14C-most-probable-number method for enumeration of active heterotrophic microorganisms in natural waters.

A most-probable-number method using 14C-labeled substrates is described for the enumeration of aquatic populations of heterotrophic microorganisms. Natural populations of microorganisms are inoculated into dilution replicates prepared from the natural water from which the organisms originated. The natural water is supplemented with a 14C-labeled compound added so as to approximate a true environmental concentration. 14CO2 evolved by individual replicates is trapped in NaOH and counted by liquid scintillation techniques for use in scoring replicates as positive or negative. Positives (14CO2 evolution) are easily distinguished from negatives (no 14CO2 evolution). The results from a variety of environments using the 14CO2 procedure agreed well with previously described methods, in most instances. The 14C-most-probable-number method described here reduces handling procedures over previously described most-probable-number procedures using 14C-labeled substrates. It also appears to have advantages over other enumeration methods in its attempt to approximate natural conditions more closely.     

Study of lignin biotransformation by Aspergillus fumigatus and white-rot fungi using (14)C-labeled and unlabeled kraft lignins

The biodegradation of lignin by fungi was studied in shake flasks using (14)C-labeled kraft lignin and in a deep-tank fermentor using unlabeled kraft lignin. Among the fungi screened, A. fumigatus-isolated in our laboratories-was most potent in lignin biotransformation. Dialysis-type fermentation, designed to study possible accumulation of low MW lignin-derived products, showed no such accumulation. Recalcitrant carbohydrates like mi-crocrystalline cellulose supported higher lignolytic activity than easily metabolized carbohydrates like cellobiose. An assay developed to distinguish between CO(2) evolved from lignin and carbohydrate substrates demonstrated no stoichiometric correlation between the metabolism of the two cosubstrates. The submerged fermentations with unlabeled lignin are difficult to monitor since chemical assays do not give accurate and true results. Lignolytic efficiencies that allowed monitoring of such fermentations were defined. Degraded lignins were analyzed for structural modifications. A. fumigatus was clearly superior to C. versicolor in all aspects of lignin degradation; A. fumigatus brought about substantial demethoxylation and dehydroxylation, whereas C. versicolor degraded lignins closely resembled undegraded kraft lignin. There was a good agreement among the different indices of lignin degradation, namely, (14)CO evolution, OCH(3) loss, OH loss, and monomer and dimer yield after permanganate oxidation.     

Method for Radiorespirometric Detection of Bacteria in Pure Culture and in Blood

Methods are described for the detection of low numbers of bacteria by monitoring (14)CO(2) evolved from (14)C-labeled substrates. Cell suspensions are filtered with membrane filters, and the filter is then moistened with 0.1 ml of labeled medium in a small, closed apparatus. Evolved (14)CO(2) is collected with Ba(OH)(2)-moistened filter pads and assayed with conventional radioactivity counting equipment. The kinetics of (14)CO(2) evolution are shown for several species of bacteria. Fewer than 100 colony-forming units of most species tested were detected in 2 h or less. Bacteria were inoculated into blood and the mixture was treated to lyse the blood cells. The suspension ws filtered and the filter was placed in a small volume of labeled medium. The evolved (14)CO(2) was trapped and counted. A key development in the methodology was finding that an aqueous solution of Rhyozyme and Triton X-100 produced lysis of blood but was not detrimental to bacteria.     

Development of a carbon dioxide-capture assay in microtiter plate for aspartyl-beta-hydroxylase.

CO2-capture methods have been used for assaying many decarboxylating enzymes including hydroxylation-coupled decarboxylation reactions. The traditional CO2-capture method involves performing the reaction in capped tubes and radiometric measurement of trapped 14CO2 by scintillation counting. In this report, a 14CO2-capture method in a 96-well microtiter plate format has been developed and a phosphor imaging system has been employed for sample measurement. The new assay method has been used successfully to assay aspartyl-beta-hydroxylase activity in microtiter plate format. The results obtained here compare favorably with those obtained from the traditional tube method. The method is sensitive, suitable for high throughput, and generally applicable to many CO2-releasing enzyme assays.     

A rapid method for detecting bacterial contamination in the presence of Penicillium and Streptomyces antibiotic fermentations

Contamination in antibiotic fermentation processes results in major economic and process problems. The detection and elimination of contamination is a continual objective for fermentation companies. While efforts continue to eliminate contamination by improving equipment and sterile techniques, it is still imperative to have a rapid method for detecting contamination in laboratory-stage inoculum and seed tanks. This article describes the successful studies leading to the adoption of the BACTEC, an automatic bacterial detection system, as a supplemental detection technique. The BACTEC system detects contamination by incubating samples with a selected(14)C-labeled substrate or substrates. The resulting metabolism of substrate produces (14)C-labeled CO(2) which is then quantified and expressed as a growth index, permitting detection of contamination more rapidly at a much earlier time than is possible with conventional detection techniques that involve Phenol red dextrose broth, streak plates, and microscopic examination techniques.     

Measurement of 14CO2 evolution during tissue oxidation in vitro; an alternative to the Kontes well

1. An alternative method to the use of the disposable Kontes well for trapping 14CO2 produced in the course of biological oxidations is described. 2. A polyethylene miniature scintillation vial was used to contain the hyamine hydroxide-impregnated filter paper wick. 3. The two methods are compared in their abilities to trap 14CO2 produced directly by acidification of sodium [14C]bicarbonate and during beta-oxidation of 1[14C] palmitic acid. 4. The miniature vial and Kontes well methods showed similar efficiencies in the trapping of 14CO2 (97% and 95%, respectively, on average) the radioactivity of which was determined in the miniature vial using 5 ml only of scintillation fluid compared with a minimum of 10 ml required by the standard scintillation vial used to accommodate the Kontes well. 5. The technical advantages of the suggested miniature vial system, during both incubation and counting stages, are discussed.     

Bacterial growth kinetics: modelling and evaluation of two-compartment radioassay

Quantitative measurements of bacterial growth may be made using a radioassay technique. This method measures, by scintillation counting, the 14CO2 derived from the bacterial metabolism of a 14C-labeled substrate. Mathematical growth models may serve as reliable tools for estimation of the generation rate constant (or slope of the growth curve) and provide a basis for evaluating assay performance. Two models, i.e., exponential and logistic, are proposed. Both models yielded an accurate fit to the data from radioactive measurement of bacterial growth. The exponential model yielded high precision values of the generation rate constant, with an average relative standard deviation of 1.2%. Under most conditions the assay demonstrated no changes in the slopes of growth curves when the number of bacteria per inoculation was changed. However, the radiometric assay by scintillation method had a growth-inhibiting effect on a few strains of bacteria. The source of this problem was thought to be hypersensitivity to trace amounts of toluene remaining on the detector.     

Carbon Dioxide Fixation in Neuronal and Astroglial Cells in Culture

The concentration of potassium in the extracellular fluid has been found to stimulate the rate of CO2 fixation by astroglial cells grown in primary culture. Raising the concentration of extracellular potassium increased both the initial rate of formation of the 14C-labeled products of 14CO2 fixation and the final steady-state level of these products within the cells. In contrast, neither veratridine nor L-glutamate affected the rate of CO2 fixation in astroglial cells. The very low rate of CO2 fixation found in primarily neuronal cultures was unaffected by increased extracellular potassium as was CO2 fixation in fibroblasts. When cultured alone, astroglial cells release a large fraction of the 14C-labeled products of CO2 fixation into the surrounding medium. Mixed cultures of astroglia and neurons also fix CO2 but, in contrast to astroglia cultured alone, release only a small fraction of the 14C-labeled products into the culture medium.     

Radiorespirometry and metabolism of cultured cells attached to petri dishes

A radiorespirometer was constructed for continuous quantitation of ¹⁴CO₂ released from specifically labeled substrates by intact cultured cells attached to plastic petri dishes. An airtight chamber is created by sealing the petri dish with a specially designed cover inside a thermostated holder. Rapid equilibration of released ¹⁴CO₂ with a 5% $\text{CO}{}_2\text{95\%}$ air carrier gas is achieved by bubbling the carrier gas under the surface of the growth medium. Labeled CO₂ is removed from the carrier gas by trapping in an organic base and quantitated by liquid scintillation counting. Additions to or sampling of the growth medium may be performed during a run and the carrier gas may be modified to test the effects of anesthetics and different O₂ levels. The ability to continuously monitor ¹⁴CO₂ release can provide valuable information concerning the metabolic pathways of substrate oxidation which cannot be obtained from single ¹⁴CO₂ determinations. A capacity of 12 culture plates enormously increases the amount of data that can be collected in a given time. The use of liquid scintillation counting increases the sensitivity and resolution over the ion chamber and Geiger counter methods, and permits utilization of the procedure in a much wider range of laboratories. Data obtained for the oxidation of specifically ¹⁴C-labeled glucose and pyruvate by neonatal rat heart cells in culture, in both the presence and absence of oxygen, are provided as examples of the utility of the method.     

Improvements in and Environmental Applications of Double-Vial Radiorespirometry for the Study of Microbial Mineralization

Several variations in the scintillation mixture and the filter paper arrangements for double-vial radiorespirometry were compared. Improved efficiencies (44%) and shorter response times were found by adding wetting agents and methanolic NaOH to the scintillation mixture in the filter paper. The scintillation chemicals used did not contain dioxane and were found to be nontoxic to the test microbiota in this system. Covering the inner reaction vial with aluminum foil minimized the reduction in counting efficiency when testing colored or dense environmental samples. Mineralization rates were determined with ¹⁴C-labeled glucose, acetate, and glutamate and [¹⁴C]cellulose- and [¹⁴C]lignin-labeled lignocellulose for composting cow manure, forest soil, and arctic lake sediment microbiota. This improved method can be used in a variety of procedures involving the measurement of microbial mineralizations of organic compounds. Since no liquid scintillation cocktail is used for counting, the radioactive wastes are aqueous or can be incinerated, making disposal easy.     

Transformation of toluene and benzene by mixed methanogenic cultures

The aromatic hydrocarbons toluene and benzene were anaerobically transformed by mixed methanogenic cultures derived from ferulic acid-degrading sewage sludge enrichments. In most experiments, toluene or benzene was the only semicontinuously supplied carbon and energy source in the defined mineral medium. No exogenous electron acceptors other than CO2 were present. The cultures were fed 1.5 to 30 mM unlabeled or 14C-labeled aromatic substrates (ring-labeled toluene and benzene or methyl-labeled toluene). Gas production from unlabeled substrates and 14C activity distribution in products from the labeled substrates were monitored over a period of 60 days. At least 50% of the substrates were converted to CO2 and methane (greater than 60%). A high percentage of 14CO2 was recovered from the methyl group-labeled toluene, suggesting nearly complete conversion of the methyl group to CO2 and not to methane. However, a low percentage of 14CO2 was produced from ring-labeled toluene or from benzene, indicating incomplete conversion of the ring carbon to CO2. Anaerobic transformation pathways for unlabeled toluene and benzene were studied with the help of gas chromatography-mass spectrometry. The intermediates detected are consistent with both toluene and benzene degradation via initial oxidation by ring hydroxylation or methyl oxidation (toluene), which would result in the production of phenol, cresols, or aromatic alcohol. Additional reactions, such as demethylation and ring reduction, are also possible. Tentative transformation sequences based upon the intermediates detected are discussed.     

Calculation of lipolysis and esterification from glycerol metabolism in rat adipose tissue

Pieces of epididymal fat pad from fed and 48-hr-fasted rats were incubated for various periods of time in Krebs-Ringer bicarbonate containing [1-(14)C]glycerol. The radioactive substrate taken up by the tissue increased linearly with time in both groups and was mainly converted to glyceride-glycerol and CO(2). The slopes of the regressions of (14)C-labeled glyceride-glycerol with time were not different between the groups, while those of (14)CO(2) were smaller in the fasted than in the fed animals. Because the radioactive glycerol in the medium is being continuously diluted with the glycerol coming out of the tissue, it is necessary to take account of this factor in calculating the actual amount of glycerol utilized by the tissue. The glycerol produced by the tissues is higher in the fasted than in the fed animals, and in both groups it increases hyperbolically with time. As negligible amounts of the (14)C-labeled glycerol taken up by the tissue recirculates to the medium, the rates of glycerol release (lipolysis), esterification, and oxidation to CO(2) were calculated.     

Transformation of toluene and benzene by mixed methanogenic cultures.

The aromatic hydrocarbons toluene and benzene were anaerobically transformed by mixed methanogenic cultures derived from ferulic acid-degrading sewage sludge enrichments. In most experiments, toluene or benzene was the only semicontinuously supplied carbon and energy source in the defined mineral medium. No exogenous electron acceptors other than CO2 were present. The cultures were fed 1.5 to 30 mM unlabeled or 14C-labeled aromatic substrates (ring-labeled toluene and benzene or methyl-labeled toluene). Gas production from unlabeled substrates and 14C activity distribution in products from the labeled substrates were monitored over a period of 60 days. At least 50% of the substrates were converted to CO2 and methane (greater than 60%). A high percentage of 14CO2 was recovered from the methyl group-labeled toluene, suggesting nearly complete conversion of the methyl group to CO2 and not to methane. However, a low percentage of 14CO2 was produced from ring-labeled toluene or from benzene, indicating incomplete conversion of the ring carbon to CO2. Anaerobic transformation pathways for unlabeled toluene and benzene were studied with the help of gas chromatography-mass spectrometry. The intermediates detected are consistent with both toluene and benzene degradation via initial oxidation by ring hydroxylation or methyl oxidation (toluene), which would result in the production of phenol, cresols, or aromatic alcohol. Additional reactions, such as demethylation and ring reduction, are also possible. Tentative transformation sequences based upon the intermediates detected are discussed.     

One carbon metabolism in SAR11 pelagic marine bacteria

The SAR11 Alphaproteobacteria are the most abundant heterotrophs in the oceans and are believed to play a major role in mineralizing marine dissolved organic carbon. Their genomes are among the smallest known for free-living heterotrophic cells, raising questions about how they successfully utilize complex organic matter with a limited metabolic repertoire. Here we show that conserved genes in SAR11 subgroup Ia (Candidatus Pelagibacter ubique) genomes encode pathways for the oxidation of a variety of one-carbon compounds and methyl functional groups from methylated compounds. These pathways were predicted to produce energy by tetrahydrofolate (THF)-mediated oxidation, but not to support the net assimilation of biomass from C1 compounds. Measurements of cellular ATP content and the oxidation of (14)C-labeled compounds to (14)CO(2) indicated that methanol, formaldehyde, methylamine, and methyl groups from glycine betaine (GBT), trimethylamine (TMA), trimethylamine N-oxide (TMAO), and dimethylsulfoniopropionate (DMSP) were oxidized by axenic cultures of the SAR11 strain Ca. P. ubique HTCC1062. Analyses of metagenomic data showed that genes for C1 metabolism occur at a high frequency in natural SAR11 populations. In short term incubations, natural communities of Sargasso Sea microbial plankton expressed a potential for the oxidation of (14)C-labeled formate, formaldehyde, methanol and TMAO that was similar to cultured SAR11 cells and, like cultured SAR11 cells, incorporated a much larger percentage of pyruvate and glucose (27-35%) than of C1 compounds (2-6%) into biomass. Collectively, these genomic, cellular and environmental data show a surprising capacity for demethylation and C1 oxidation in SAR11 cultures and in natural microbial communities dominated by SAR11, and support the conclusion that C1 oxidation might be a significant conduit by which dissolved organic carbon is recycled to CO(2) in the upper ocean.     

An accurate and sensitive assay of [14C]octanoate oxidation and its application on tissue homogenates and fibroblasts

A procedure was developed to assay [14C]octanoate oxidation from the production of both 14CO2 and 14C-labeled acid-soluble products. Octanoic acid and its CoA and carnitine esters were eliminated from the acid-soluble products by alkaline hydrolysis of the esters and acidification and binding of the acid to Lipidex 1000. The method was evaluated with homogenates of various rat tissues and human muscles and with human fibroblasts. 14CO2 production was variable and comprised less than 3% of the total oxidation products with homogenates and 26 +/- 19% with fibroblasts. As compared to palmitate, oxidation rates of octanoate were higher in rat liver and heart homogenates, of the same magnitude in muscle homogenates, but lower in fibroblasts. The proportion of antimycin-insensitive oxidation was much lower with octanoate than with palmitate. Using the assay a case of medium-chain acyl-CoA dehydrogenase deficiency could be indicated.     

Inhibition of CO2 production from aminopyrine or methanol by cyanamide or crotonaldehyde and the role of mitochondrial aldehyde dehydrogenase in formaldehyde oxidation

Previous results have shown that cyanamide or crotonaldehyde are effective inhibitors of the oxidation of formaldehyde by the low-Km mitochondrial aldehyde dehydrogenase, but do not affect the activity of the glutathione-dependent formaldehyde dehydrogenase. These compounds were used to evaluate the enzyme pathways responsible for the oxidation of formaldehyde generated during the metabolism of aminopyrine or methanol by isolated hepatocytes. Both cyanamide and crotonaldehyde inhibited the production of 14CO2 from 14C-labeled aminopyrine by 30-40%. These agents caused an accumulation of formaldehyde which was identical to the loss in CO2 production, indicating that the inhibition of CO2 production reflected an inhibition of formaldehyde oxidation. The oxidation of methanol was stimulated by the addition of glyoxylic acid, which increases the rate of H2O2 generation. Crotonaldehyde inhibited CO2 production from methanol, but caused a corresponding increase in formaldehyde accumulation. The partial sensitivity of CO2 production to inhibition by cyanamide or crotonaldehyde suggests that both the mitochondrial aldehyde dehydrogenase and formaldehyde dehydrogenase contribute towards the metabolism of formaldehyde which is generated from mixed-function oxidase activity or from methanol, just as both enzyme systems contribute towards the metabolism of exogenously added formaldehyde.     

Rapid Method for the Radioisotopic Analysis of Gaseous End Products of Anaerobic Metabolism

A gas chromatographic procedure for the simultaneous analysis of (14)C-labeled and unlabeled metabolic gases from microbial methanogenic systems is described. H(2), CH(4), and CO(2) were separated within 2.5 min on a Carbosieve B column and were detected by thermal conductivity. Detector effluents were channeled into a gas proportional counter for measurement of radioactivity. This method was more rapid, sensitive, and convenient than gas chromatography-liquid scintillation techniques. The gas chromatography-gas proportional counting procedure was used to characterize the microbial decomposition of organic matter in anaerobic lake sediments and to monitor (14)CH(4) formation from H(2) and (14)CO(2) by Methanosarcina barkeri.