13C-methacetin breath test parameter S for liver diseases diagnosis

The mechanism of ¹³C-methacetin breath test is set forth clearly with the analysis of pharmacokinetics mode, and the measuring method of ¹³C-methacetin breath test and its clinical applications in the diagnosis of liver diseases are reported in detail. On the basis of comprehensive analysis of the clinical test data, the advanced diagnostic parameter S is of important significance for the application and development of breath test.     

On-line measurements of C enrichments in rat breath: Non-invasive method for in vivo study of drug enzymatic induction

A differential kinetic study of ¹³CO₂ enrichment of breath after the intake of specific ¹³C-labelled substrates and co-administration of a drug allows the drug's ability for enzyme induction to be evaluated in vivo. A method and a gas chromatograph—isotope ratio mass spectrometer device for on-line measurements of ¹³CO₂ enrichment in the breath of small animals are described. This system allows on-line breath sample collection from a metabolic cage, purification by gas chromatography, determination of CO₂ by thermal conductivity detection and measurement of ¹³CO₂ enrichment by isotope ratio mass spectrometry. Two protocols for phenobarbital-inducible P450 and 3-methylcholanthrene-inducible P1-450 isoenzymes are described.     

Isotope Labeling and Microautoradiography of Active Heterotrophic Bacteria on the Basis of Assimilation of 14CO2

Most heterotrophic bacteria assimilate CO₂ in various carboxylation reactions during biosynthesis. In this study, assimilation of ¹⁴CO₂ by heterotrophic bacteria was used for isotope labeling of active microorganisms in pure cultures and environmental samples. Labeled cells were visualized by microautoradiography (MAR) combined with fluorescence in situ hybridization (FISH) to obtain simultaneous information about activity and identity. Cultures of Escherichia coli and Pseudomonas putida assimilated sufficient ¹⁴CO₂ during growth on various organic substrates to obtain positive MAR signals. The MAR signals were comparable with the traditional MAR approach based on uptake of ¹⁴C-labeled organic substrates. Experiments with E. coli showed that ¹⁴CO₂ was assimilated during both fermentation and aerobic and anaerobic respiration. The new MAR approach, HetCO₂-MAR, was evaluated by targeting metabolic active filamentous bacteria, including “Candidatus Microthrix parvicella” in activated sludge. “Ca. Microthrix parvicella” was able to take up oleic acid under anaerobic conditions, as shown by the traditional MAR approach with [¹⁴C]oleic acid. However, the new HetCO₂-MAR approach indicated that “Ca. Microthrix parvicella,” did not significantly grow on oleic acid under anaerobic conditions with or without addition of NO₂⁻, whereas the addition of O₂ or NO₃⁻ initiated growth, as indicated by detectable ¹⁴CO₂ assimilation. This is a metabolic feature that has not been described previously for filamentous bacteria. Such information could not have been derived by using the traditional MAR procedure, whereas the new HetCO₂-MAR approach differentiates better between substrate uptake and substrate metabolism that result in growth. The HetCO₂-MAR results were supported by stable isotope analysis of ¹³C-labeled phospholipid fatty acids from activated sludge incubated under aerobic and anaerobic conditions in the presence of ¹³CO₂. In conclusion, the novel HetCO₂-MAR approach expands the possibility for studies of the ecophysiology of uncultivated microorganisms.     

Does the Diagnostic Accuracy of the 13C‐Urea Breath Test Vary with Age Even After the Application of Urea Hydrolysis Rate?

Background: Endogenous CO₂ production may be a possible explanation for higher false‐positive results reported for ¹³C‐urea breath test (UBT) in children below 6 years. In this study, we evaluated whether age affects the diagnostic accuracy of the ¹³C‐UBT even after the application of urea hydrolysis rate (UHR) in children. Methods: A total of 612 ¹³C‐UBTs and endoscopic biopsies were performed on children divided into two groups; children under 6 years (n = 126) and children aged 6–18 years (n = 486). For ¹³C‐UBT, 75 mg ¹³C‐urea was ingested, and breath sample was collected 30 minutes later. Delta over baseline (DOB) was determined, and UHR was calculated to normalize the DOB values for endogenous CO₂ production. Results: There was significant difference between the DOB values of children under 6 years and those of children over 6 years in H. pylori‐positive (p = .029) and ‐negative groups (p = .002). On applying the UHR, no significant difference was observed between the UHR values of children under 6 years and those of children over 6 years in H. pylori‐positive (p = .877) and ‐negative groups (p = .427). In 12.6% children under 6 years, false‐positive results were observed on applying the DOB, and in 9.0% on applying the UHR (p = .125). Conclusions: The ¹³C‐UBT is a noninvasive method exhibiting high diagnostic accuracy with both UHR as well as DOB. However, high false‐positive results for ¹³C‐UBT were noted in children below 6 years on applying both UHR as well as DOB. Thus, this may not only be due to the effects of endogenous CO₂ production but also due to other factors.     

Functional activity of the liver under the conditions of immersion and effects of countermeasures

Ultrasound investigations (USI) of the liver, organs and vessels of the gastroduodenal area, as well as blood biochemistry, were performed in two groups of male volunteers on the 4th day of their stay in the conditions of “dry” immersion with and without the application of countermeasures, including the support load imitator (SLI) or high-frequency electromyostimulation. Using ¹³С-methacetin breath test (¹³C-MBT), two other groups were investigated for the effect of immersion on the detoxification activity and metabolic capacity of the liver and the efficacy of SLI. The performed USIs have identified deceleration in the hepatic venous flow and the signs of plethora in the abdominal venous system. Elevated blood levels were detected in pepsinogen, pancreatic amylase, bilirubin total, due to its unconjugated fraction, insulin, and C-peptide. The ¹³C-MBT has shown a slowdown in the rate of ¹³C-methacetin inactivation and a reduction in the hepatic metabolic capacity. The application of countermeasures during the immersion has not affected the ultrasound patterns of the hemodynamic rearrangement in both the liver and the abdomen. High frequency electromyostimulation during the immersion has neutralized the changes in all biochemical indicators except C-peptide, while the application of SLI has led to the restoration of only pepsinogen and amylase to the initial values. In addition, the use of SLI during the immersion counteracted the reduction in the ¹³C-methacetin inactivation rate and did not substantially affect the reduction in the metabolic capacity of the liver.     

13[C]-Urea Breath Test as a Novel Point-of-Care Biomarker for Tuberculosis Treatment and Diagnosis

Background

Pathogen-specific metabolic pathways may be detected by breath tests based on introduction of stable isotopically-labeled substrates and detection of labeled products in exhaled breath using portable infrared spectrometers.

Methodology/Principal Findings

We tested whether mycobacterial urease activity could be utilized in such a breath test format as the basis of a novel biomarker and diagnostic for pulmonary TB. Sensitized New-Zealand White Rabbits underwent bronchoscopic infection with either Mycobacterium bovis or Mycobacterium tuberculosis. Rabbits were treated with 25 mg/kg of isoniazid (INH) approximately 2 months after infection when significant cavitary lung pathology was present. [¹³C] urea was instilled directly into the lungs of intubated rabbits at selected time points, exhaled air samples analyzed, and the kinetics of δ¹³CO₂ formation were determined. Samples obtained prior to inoculation served as control samples for background ¹³CO₂ conversion in the rabbit model. ¹³CO₂, from metabolic conversion of [¹³C]-urea by mycobacterial urease activity, was readily detectable in the exhaled breath of infected rabbits within 15 minutes of administration. Analyses showed a rapid increase in the rate of ¹³CO₂ formation both early in disease and prior to treatment with INH. Following INH treatment, all evaluable rabbits showed a decrease in the rate of ¹³CO₂ formation.

Conclusions/Significance

Urea breath testing may provide a useful diagnostic and biomarker assay for tuberculosis and for treatment response. Future work will test specificity for M. tuberculosis using lung-targeted dry powder inhalation formulations, combined with co-administering oral urease inhibitors together with a saturating oral dose of unlabeled urea, which would prevent the δ¹³CO₂ signal from urease-positive gastrointestinal organisms.     

New Insights into the Ligninolytic Capability of a Wood Decay Ascomycete

Wood-grown cultures of Daldinia concentrica oxidized a permethylated β-¹⁴C-labeled synthetic lignin to ¹⁴CO₂ and also cleaved a permethylated α-¹³C-labeled synthetic lignin to give C_α-C_β cleavage products that were detected by ¹³C nuclear magnetic resonance spectrometry. Therefore, this ascomycete resembles white-rot basidiomycetes in attacking the recalcitrant nonphenolic structures that predominate in lignin.     

Forms of Dissolved Carbon Dioxide Required for Photosystem II Activity in Chloroplast Membranes

High concentrations of both bicarbonate and formate inhibit photosynthetic O₂ evolution at pH 8.0. At this pH, only 2.4% of the total dissolved carbon dioxide exists as CO₂. At pH 7.3, where 11% of the total dissolved carbon dioxide exists as CO₂, HCO₃⁻ no longer inhibits. While formate still inhibits O₂ evolution at pH 7.3, its effect can be partially overcome if CO₂ is also present. The rate of binding of added ¹⁴C-labeled inorganic carbon is nearly 10-fold more rapid when the internal pH of thylakoid membranes is at 6.0 than when it is at 7.8. These observations suggest that CO₂, not HCO₃⁻, is initially bound to the photosystem II reaction center and that the location of the binding site is on the inside thylakoid surface. However, additional data presented here suggest that, after binding, CO₂ is hydrated to HCO₃⁻ + H⁺ in a pH-dependent reaction. Two possible explanations of the “bicarbonate effect” are presented.     

Partitioning of CH4 and CO2 Production Originating from Rice Straw,Soil and Root Organic Carbon in Rice Microcosms

Flooded rice fields are an important source of the greenhouse gas CH₄. Possible carbon sources for CH₄ and CO₂ production in rice fields are soil organic matter (SOM), root organic carbon (ROC) and rice straw (RS), but partitioning of the flux between the different carbon sources is difficult. We conducted greenhouse experiments using soil microcosms planted with rice. The soil was amended with and without ¹³C-labeled RS, using two ¹³C-labeled RS treatments with equal RS (5 g kg⁻¹ soil) but different δ¹³C of RS. This procedure allowed to determine the carbon flux from each of the three sources (SOM, ROC, RS) by determining the δ¹³C of CH₄ and CO₂ in the different incubations and from the δ¹³C of RS. Partitioning of carbon flux indicated that the contribution of ROC to CH₄ production was 41% at tillering stage, increased with rice growth and was about 60% from the booting stage onwards. The contribution of ROC to CO₂ was 43% at tillering stage, increased to around 70% at booting stage and stayed relatively constant afterwards. The contribution of RS was determined to be in a range of 12–24% for CH₄ production and 11–31% for CO₂ production; while the contribution of SOM was calculated to be 23–35% for CH₄ production and 13–26% for CO₂ production. The results indicate that ROC was the major source of CH₄ though RS application greatly enhanced production and emission of CH₄ in rice field soil. Our results also suggest that data of CH₄ dissolved in rice field could be used as a proxy for the produced CH₄ after tillering stage.     

Microalgae as a source of stable isotopically labeled compounds

Microalgae have the ability to convert inorganic compounds into organic compounds. When they are cultured in the presence of stable (non-radioactive) isotopes (i.e.¹³CO₂,¹⁵NO ₃ ^– ,²H₂O) their biomass becomes labeled with the stable isotopes, and a variety of stable isotopically-labeled compounds can be extracted and purified from that biomass.Two applications for stable isotopically-labeled compounds are as cell culture nutrients and as breath test diagnostics. Bacteria that are cultured with labeled nutrients will produce bacterial products that are labeled with stable isotopes. The presence of these isotopes in the bacterial products, along with recent developments in NMR technology, greatly reduces the time and effort required to determine the three-dimensional structure of macromolecules and the interaction of proteins with ligands. As breath test diagnostics, compounds labeled with¹³C are used to measure the metabolism of particular organs and thus diagnose various disease conditions. These tests are based on the principle that a particular compound is metabolized primarily by a single organ, and when that compound is labeled with¹³C, the appearance of¹³CO₂ in exhaled breath provides information about the metabolic activity of the target organ. Tests of this type are simple to perform, non-invasive, and less expensive than many conventional diagnostic procedures.The commercialization of stable isotopically labeled compounds requires that these compounds be produced in a cost-effective manner. Our approach is to identify microalgal overproducers of the desired compounds, maximize the product content of those organisms, and purify the resulting products.     

Change in the peripheral CO2 chemoreflex from rest to exercise

A single-breath CO₂ test of peripheral chemosensitivity has recently been described, and elaborated based on model simulations. This study was designed to measure the peripheral CO₂ chemoreflex at rest and during heavy exercise to see if carotid chemosensitivity to CO₂ increased. Ten healthy, adult males performed an incremental exercise test to determine their ventilatory anaerobic threshold (VAT), and 20 minutes of steady-state exercise at a pre-determined power output above VAT. Arterialized venous blood was obtained during each minute of incremental exercise to verify development of metabolic acidosis. Carotid chemosensitivity was tested repeatedly at rest and in steady-state exercise by the ventilatory response to a single breath of 13% CO₂ in air. The peripheral chemoreflex for CO₂ for the group of subjects doubled from rest to exercise (mean 0.0961 · s^–1 · kPa^–1) with all subjects showing an increase. We conclude that the gain of the carotid CO₂ chemoreflex increases from rest to exercise at work above the VAT.     

Terpene Biosynthesis in Cell-free Extracts and Excised Shoots from Wedgwood Iris

Excised shoots and cell-free extracts prepared from Wedgwood iris (Iris hollandica Hoog. “Wedgwood”) shoots metabolized ¹⁴C-labeled mevalonic acid (MVA). By using cell-free extracts, the ¹⁴C from MVA-1-¹⁴C was recovered as ¹⁴CO₂, while that from MVA-2-¹⁴C was recovered as neutral terpenes, acid-hydrolyzable terpenes, or ¹⁴CO₂. Also, under optimal incubation conditions, 12.8 nanomoles R-MVA-2-¹⁴C was incorporated into neutral terpenes per milligram fresh weight per hour. In contrast, excised shoots incorporated only 0.58 nanomoles R-MVA-2-¹⁴C per mg fresh weight per hour. Labeled products identified from the cell-free system were squalene, farnesol, geranylgeraniol, and compounds that are converted to farnesol and geranylgeraniol after alkaline hydrolysis. Squalene and a 4,4-dimethylsterol were identified as products from excised shoots but not the terpene alcohols or the alkaline-hydrolyzable compounds.     

Role of Carbon Dioxide in Catabolism of Propane by “Nocardia paraffinicum” (Rhodococcus rhodochrous)

The catabolism of propane by “Nocardia paraffinicum” (Rhodococcus rhodochrous) has been shown to involve CO₂ fixation after its oxidation to propionic acid. “N. paraffinicum” failed to grow on either propane or 1-propanol in the absence of CO₂. The rate of propane utilization was directly related to the initial CO₂ concentration, and Warburg respirometry suggested that CO₂ was required for the catabolism of 1-propanol, propionaldehyde, and propionate but not for 2-propanol. These data also suggested that the predominant pathway for the utilization of propane by “N. paraffinicum” was through 1-propanol. The use of [2-¹⁴C]propane and ¹⁴CO₂ confirmed the catabolism of propane and the fixation of CO₂. Through the use of these isotopes and the pyruvate carboxylase inhibitor sodium arsenite, the labeled 2,4-dinitrophenylhydrazine derivative of pyruvate was trapped and isolated via thin-layer chromatography. The trapping of [¹⁴C]pyruvate in this manner was considered to be indicative of the presence of the methylmalonyl coenzyme A pathway for CO₂ fixation.     

The Rate of Photorespiration during Photosynthesis and the Relationship of the Substrate of Light Respiration to the Products of Photosynthesis in Sunflower Leaves

Single attached leaves of sunflower (Helianthus annus L. “Mennonite”) were supplied ¹⁴CO₂ of constant specific radioactivity in gas mixtures containing various CO₂ and O₂ concentrations. The ¹⁴CO₂ and CO₂ fluxes were measured concurrently in an open system using an ionization chamber and infrared gas analyzer.     

Characterization and In Situ Carbon Metabolism of Phototrophic Consortia

A dense population of the phototrophic consortium “Pelochromatium roseum” was investigated in the chemocline of a temperate holomictic lake (Lake Dagow, Brandenburg, Germany). Fluorescence in situ hybridization revealed that the brown epibionts of “P. roseum” constituted up to 37% of the total bacterial cell number and up to 88% of all green sulfur bacteria present in the chemocline. Specific amplification of 16S rRNA gene fragments of green sulfur bacteria and denaturing gradient gel electrophoresis fingerprinting yielded a maximum of four different DNA bands depending on the year of study, indicating that the diversity of green sulfur bacteria was low. The 465-bp 16S rRNA gene sequence of the epibiont of “P. roseum” was obtained after sorting of individual consortia by micromanipulation, followed by a highly sensitive PCR. The sequence obtained represents a new phylotype within the radiation of green sulfur bacteria. Maximum light-dependent H¹⁴CO₃⁻ fixation in the chemocline in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea suggested that there was anaerobic autotrophic growth of the green sulfur bacteria. The metabolism of the epibionts was further studied by determining stable carbon isotope ratios (δ¹³C) of their specific biomarkers. Analysis of photosynthetic pigments by high-performance liquid chromatography revealed the presence of high concentrations of bacteriochlorophyll (BChl) e and smaller amounts of BChl a and d and chlorophyll a in the chemocline. Unexpectedly, isorenieratene and β-isorenieratene, carotenoids typical of other brown members of the green sulfur bacteria, were absent. Instead, four different esterifying alcohols of BChl e were isolated as biomarkers of green sulfur bacterial epibionts, and their δ¹³C values were determined. Farnesol, tetradecanol, hexadecanol, and hexadecenol all were significantly enriched in ¹³C compared to bulk dissolved and particulate organic carbon and compared to the biomarkers of purple sulfur bacteria. The difference between the δ¹³C values of farnesol, the major esterifying alcohol of BChl e, and CO₂ was −7.1%, which provides clear evidence that the mode of growth of the green sulfur bacterial epibionts of “P. roseum” in situ is photoautotrophic.     

ManR,a Transcriptional Regulator of the β-Mannan Utilization System,Controls the Cellulose Utilization System in Aspergillus oryzae

The author modified a respiratory gas analyzer to analyze the respiratory ¹³CO₂ of 12 small laboratory animals all at once. To investigate the practical use of this system, mice were orally (OR) or intravenously (IV) given glucose solutions containing three different amounts of ¹³C-labeled glucose. Expired ¹³CO₂ derived from exogenous glucose was detected within 10 minutes after administration in OR mice, but about 30 minutes in IV mice. The height of the peak of ¹³CO₂ expiration was correlated with the administered ¹³C-glucose mass.     

Utilization of Exogenous Inorganic Carbon Species in Photosynthesis by Chlorella pyrenoidosa

The nature of the inorganic carbon utilized during photosynthesis by Chlorella pyrenoidosa was investigated using three experimental techniques (open gas analysis system with “artificial leaf” or “aqueous” chambers and O₂ electrode system) to measure carbon assimilation. Photosynthesis was studied as a function of pH and CO₂ concentration. The CO₂ concentration was inadequate to meet the requirements of photosynthesis only when HCO₃⁻ was added at high pH. Under all other conditions, the low and constant K_m (CO₂), in contrast to the highly variable K_m (HCO₃⁻), suggested that CO₂ was the major species utilized.     

Stable-Isotope Probing of Microorganisms Thriving at Thermodynamic Limits: Syntrophic Propionate Oxidation in Flooded Soil

Propionate is an important intermediate of the degradation of organic matter in many anoxic environments. In methanogenic environments, due to thermodynamic constraints, the oxidation of propionate requires syntrophic cooperation of propionate-fermenting proton-reducing bacteria and H₂-consuming methanogens. We have identified here microorganisms that were active in syntrophic propionate oxidation in anoxic paddy soil by rRNA-based stable-isotope probing (SIP). After 7 weeks of incubation with [¹³C]propionate (<10 mM) and the oxidation of ~30 μmol of ¹³C-labeled substrate per g dry weight of soil, we found that archaeal nucleic acids were ¹³C labeled to a larger extent than those of the bacterial partners. Nevertheless, both terminal restriction fragment length polymorphism and cloning analyses revealed Syntrophobacter spp., Smithella spp., and the novel Pelotomaculum spp. to predominate in “heavy” ¹³C-labeled bacterial rRNA, clearly showing that these were active in situ in syntrophic propionate oxidation. Among the Archaea, mostly Methanobacterium and Methanosarcina spp. and also members of the yet-uncultured “rice cluster I” lineage had incorporated substantial amounts of ¹³C label, suggesting that these methanogens were directly involved in syntrophic associations and/or thriving on the [¹³C]acetate released by the syntrophs. With this first application of SIP in an anoxic soil environment, we were able to clearly demonstrate that even guilds of microorganisms growing under thermodynamic constraints, as well as phylogenetically diverse syntrophic associations, can be identified by using SIP. This approach holds great promise for determining the structure and function relationships of further syntrophic or other nutritional associations in natural environments and for defining metabolic functions of yet-uncultivated microorganisms.