Measurement of relative lung perfusion with electrical impedance and positron emission tomography: an experimental comparative study in pigs.

BACKGROUND: Electrical impedance tomography (EIT) with indicator dilution may be clinically useful to measure relative lung perfusion, but there is limited information on the performance of this technique. METHODS: Thirteen pigs (50-66 kg) were anaesthetised and mechanically ventilated. Sequential changes in ventilation were made: (i) right-lung ventilation with left-lung collapse, (ii) two-lung ventilation with optimised PEEP, (iii) two-lung ventilation with zero PEEP after saline lung lavage, (iv) two-lung ventilation with maximum PEEP (20/25 cm H2O to achieve peak airway pressure 45 cm H2O), and (v) two-lung ventilation under unilateral pulmonary artery occlusion. Relative lung perfusion was assessed with EIT and central venous injection of saline 3%, 5%, and 10% (10 ml) during breath holds. Relative perfusion was determined by positron emission tomography (PET) using 68Gallium-labelled microspheres. EIT and PET were compared in eight regions of equal ventro-dorsal height (right, left, ventral, mid-ventral, mid-dorsal, and dorsal), and directional changes in regional perfusion were determined. RESULTS: Differences between methods were relatively small (95% of values differed by less than 8.7%, 8.9%, and 9.5% for saline 10%, 5%, and 3%, respectively). Compared with PET, EIT underestimated relative perfusion in dependent, and overestimated it in non-dependent, regions. EIT and PET detected the same direction of change in relative lung perfusion in 68.9-95.9% of measurements. CONCLUSIONS: The agreement between EIT and PET for measuring and tracking changes of relative lung perfusion was satisfactory for clinical purposes. Indicator-based EIT may prove useful for measuring pulmonary perfusion at bedside.

Variable versus conventional lung protective mechanical ventilation during open abdominal surgery (PROVAR): a randomised controlled trial.

BACKGROUND: Experimental studies showed that controlled variable ventilation (CVV) yielded better pulmonary function compared to non-variable ventilation (CNV) in injured lungs. We hypothesized that CVV improves intraoperative and postoperative respiratory function in patients undergoing open abdominal surgery. METHODS: Fifty patients planned for open abdominal surgery lasting >3 h were randomly assigned to receive either CVV or CNV. Mean tidal volumes and PEEP were set at 8 ml kg-1 (predicted body weight) and 5 cm H2O, respectively. In CVV, tidal volumes varied randomly, following a normal distribution, on a breath-by-breath basis. The primary endpoint was the forced vital capacity (FVC) on postoperative Day 1. Secondary endpoints were oxygenation, non-aerated lung volume, distribution of ventilation, and pulmonary and extrapulmonary complications until postoperative Day 5. RESULTS: FVC did not differ significantly between CVV and CNV on postoperative Day 1, 61.5 (standard deviation 22.1) % vs 61.9 (23.6) %, respectively; mean [95% confidence interval (CI)] difference, -0.4 (-13.2-14.0), P=0.95. Intraoperatively, CVV did not result in improved respiratory function, haemodynamics, or redistribution of ventilation compared to CNV. Postoperatively, FVC, forced expiratory volume at the first second (FEV1), and FEV1/FVC deteriorated, while atelectasis volume and plasma levels of interleukin-6 and interleukin-8 increased, but values did not differ between groups. The incidence of postoperative pulmonary and extrapulmonary complications was comparable in CVV and CNV. CONCLUSIONS: In patients undergoing open abdominal surgery, CVV did not improve intraoperative and postoperative respiratory function compared with CNV. CLINICAL TRIAL REGISTRATION: NCT 01683578.

Evaluation of inter-individual differences in gut bacterial isoflavone bioactivation in humans by PCR-based targeting of genes involved in equol formation.

AIM: To identify human subjects harbouring intestinal bacteria that bioactivate daidzein to equol using a targeted PCR-based approach. METHODS AND RESULTS: In a pilot study including 17 human subjects, equol formation was determined in faecal slurries. In parallel, faecal DNA was amplified by PCR using degenerate primers that target highly conserved regions of dihydrodaidzein reductase and tetrahydrodaidzein reductase genes. PCR products of the expected size were observed for six of the eight subjects identified as equol producers. Analysis of clone libraries revealed the amplification of sequences exclusively related to Adlercreutzia equolifaciens in four of the subjects tested positive for equol formation, whereas in three of the equol producers, only sequences related to Slackia isoflavoniconvertens were observed. No amplicons were obtained for one equol-forming subject, thus suggesting the presence of nontargeted alternative genes. Amplicons were only sporadically observed in the nonequol producers. CONCLUSION: The majority of human subjects who produced equol were also detected with the developed PCR-based approach. SIGNIFICANCE AND IMPACT OF THE STUDY: The obtained results shed light on the distribution and the diversity of known equol-forming bacterial species in the study group and indicate the presence of as yet unknown equol-forming bacteria.

Effects of airway tree asymmetry on the emergence and spatial persistence of ventilation defects.

Asymmetry and heterogeneity in the branching of the human bronchial tree are well documented, but their effects on bronchoconstriction and ventilation distribution in asthma are unclear. In a series of seminal studies, Venegas et al. have shown that bronchoconstriction may lead to self-organized patterns of patchy ventilation in a computational model that could explain areas of poor ventilation [ventilation defects (VDefs)] observed in positron emission tomography images during induced bronchoconstriction. To investigate effects of anatomic asymmetry on the emergence of VDefs we used the symmetric tree computational model that Venegas and Winkler developed using different trees, including an anatomic human airway tree provided by M. Tawhai (University of Auckland), a symmetric tree, and three trees with intermediate asymmetry (Venegas JG, Winkler T, Musch G, Vidal Melo MF, Layfield D, Tgavalekos N, Fischman AJ, Callahan RJ, Bellani G, Harris RS. Nature 434: 777-782, 2005 and Winkler T, Venegas JG. J Appl Physiol 103: 655-663, 2007). Ventilation patterns, lung resistance (RL), lung elastance (EL), and the entropy of the ventilation distribution were compared at different levels of airway smooth muscle activation. We found VDefs emerging in both symmetric and asymmetric trees, but VDef locations were largely persistent in asymmetric trees, and bronchoconstriction reached steady state sooner than in a symmetric tree. Interestingly, bronchoconstriction in the asymmetric tree resulted in lower RL (∼%50) and greater EL (∼%25). We found that VDefs were universally caused by airway instability, but asymmetry in airway branching led to local triggers for the self-organized patchiness in ventilation and resulted in persistent locations of VDefs. These findings help to explain the emergence and the persistence in location of VDefs found in imaging studies.

Isolation of catechin-converting human intestinal bacteria.

AIMS: To isolate and characterize bacteria from the human intestine that are involved in the conversion of catechins, a class of bioactive polyphenols abundant in the human diet. METHODS AND RESULTS: Two bacterial strains, rK3 and aK2, were isolated from an epicatechin-converting human faecal suspension. The isolates catalysed individual steps in the degradation of ⁻-epicatechin and ⁺-catechin. Based on their phenotypic characteristics and 16S rRNA gene sequences, the isolates were identified as Eggerthella lenta and Flavonifractor plautii (formerly Clostridium orbiscindens). Eggerthella lenta rK3 reductively cleaved the heterocyclic C-ring of both ⁻-epicatechin and ⁺-catechin giving rise to 1-(3,4-dihydroxyphenyl)-3-(2,4,6-trihydroxyphenyl)propan-2-ol. The conversion of catechin proceeded five times faster than that of epicatechin. Higher (epi)catechin concentrations led to an accelerated formation of the ring fission product without affecting the growth of Eg. lenta rK3. Flavonifractor plautii aK2 further converted 1-(3,4-dihydroxyphenyl)-3-(2,4,6-trihydroxyphenyl)propan-2-ol to 5-(3,4-dihydroxyphenyl)-γ-valerolactone and 4-hydroxy-5-(3,4-dihydroxyphenyl)valeric acid. Flavonifractor plautii DSM 6740 catalysed the identical reaction indicating it is not strain specific. CONCLUSIONS: The conversion of dietary catechins by the isolated Eg. lenta and F. plautii strains in the human intestine may affect their bioavailability. SIGNIFICANCE AND IMPACT OF THE STUDY: The majority of catechin metabolites are generated by the intestinal microbiota. The identification of catechin-converting gut bacteria therefore contributes to the elucidation of the bioactivation and the health effects of catechins.

[Genetic differences in enzymes of folic acid metabolism in patients with lip-jaw-palate clefts and their relatives]. / Genetische Unterschiede von Enzymen des Folsäurestoffwechsels bei Patienten mit Lippen-Kiefer-Gaumen-Spalten und ihren Angehörigen.

BACKGROUND: The effectiveness of folic acid supplementation in the periconceptional period for the prevention of cleft lip/cleft lip and palate (CLP) is contradictorily discussed. Genetically determined variants of enzymes of the folic acid metabolism could be part of the key to success or failure of folate supplementation. A mutation of the methylenetetrahydrofolate reductase (MTHFR) gene is suspected to be a risk factor for CLP. METHODS: The blood samples of 66 CLP patients, their 88 relatives (without CLP), and 184 healthy controls were searched by polymerase chain reaction for mutations of MTHFR 677 C:T, MTHFR 1298 A:C and of the arylamine N-acetyltransferase (NAT1) gene [gene type NAT1 degree 4 (wild type) or not]. RESULTS: There was no significant difference in the number of MTHFR gene mutations (for 677 C:T and 1298 A:C) between the three groups (p approximately 0.3), but for the NAT1 genes (p = 0.033). The homozygote mutation was found more than twice as often in CLP patients (10.5%) and their relatives (10.6%) than in the healthy controls (4.35%). DISCUSSION: Our results provide no evidence that the above MTHFR gene mutations are a risk factor for CLP.A NAT1 gene mutation instead could be a risk factor for CLP.

A fluorescence quenching test for the detection of flavonoid transformation.

A novel fluorescence quenching test for the detection of flavonoid degradation by microorganisms was developed. The test is based on the ability of the flavonoids to quench the fluorescence of 1,6-diphenyl-1,3,5-hexatriene (DPH). Several members of the anthocyanidins, flavones, isoflavones, flavonols, flavanones, dihydroflavanones, chalcones, dihydrochalcones and catechins were tested with regard to their quenching properties. The anthocyanidins were the most potent quenchers of DPH fluorescence, while the flavanones, dihydroflavanones and dihydrochalcones, quenched the fluorescence only weakly. The catechins had no visible impact on DPH fluorescence. The developed test allows a quick and easy differentiation between flavonoid-degrading and flavonoid-non-degrading bacteria. The investigation of individual reactions of flavonoid transformation with the developed test system is also possible.

Degradation of quercetin and luteolin by Eubacterium ramulus.

The degradation of the flavonol quercetin and the flavone luteolin by Eubacterium ramulus, a strict anaerobe of the human intestinal tract, was studied. Resting cells converted these flavonoids to 3,4-dihydroxyphenylacetic acid and 3-(3,4-dihydroxyphenyl)propionic acid, respectively. The conversion of quercetin was accompanied by the transient formation of two intermediates, one of which was identified as taxifolin based on its specific retention time and UV and mass spectra. The structure of the second intermediate, alphitonin, was additionally elucidated by (1)H and (13)C nuclear magnetic resonance analysis. In resting-cell experiments, taxifolin in turn was converted via alphitonin to 3,4-dihydroxyphenylacetic acid. Alphitonin, which was prepared by enzymatic conversion of taxifolin and subsequent purification, was also transformed to 3,4-dihydroxyphenylacetic acid. The coenzyme-independent isomerization of taxifolin to alphitonin was catalyzed by cell extract or a partially purified enzyme preparation of E. ramulus. The degradation of luteolin by resting cells of E. ramulus resulted in the formation of the intermediate eriodictyol, which was identified by high-performance liquid chromatography and mass spectrometry analysis. The observed intermediates of quercetin and luteolin conversion suggest that the degradation pathways in E. ramulus start with an analogous reduction step followed by different enzymatic reactions depending on the additional 3-hydroxyl group present in the flavonol structure.

The F420H2-dehydrogenase from Methanolobus tindarius: cloning of the ffd operon and expression of the genes in Escherichia coli.

The membrane-bound F420H2-dehydrogenase from the methylotrophic methanogen Methanolobus tindarius oxidizes reduced coenzyme F420 and feeds the electrons into an energy-conserving electron transport chain. Based on the N-terminal amino acid sequence of the 40-kDa subunit of F420H2-dehydrogenase the corresponding gene ffdB was detected in chromosomal DNA of M. tindarius. Sequence analysis, primer extension, and RT-PCR experiments indicated that ffdB is part of an operon harboring three additional open reading frames (ffdA, ffdC, ffdD). The corresponding mRNA transcript and transcription start sites were determined. All four genes could be heterologously expressed in Escherichia coli.

The sodium ion translocating glutaconyl-CoA decarboxylase from Acidaminococcus fermentans: cloning and function of the genes forming a second operon.

Glutaconyl-CoA decarboxylase from Acidaminococcus fermentans (clostridal cluster IX), a strict anaerobic inhabitant of animal intestines, uses the free energy of decarboxylation (delta G(o) approximately -30 kJ mol-1) in order to translocate Na+ from the inside through the cytoplasmic membrane. The proton, which is required for decarboxylation, most probably comes from the outside. The enzyme consists of four different subunits. The largest subunit, alpha or GcdA (65 kDa), catalyses the transfer of CO2 from glutaconyl-CoA to biotin covalently attached to the gamma-subunit, GcdC. The beta-subunit, GcdB, is responsible for the decarboxylation of carboxybiotin, which drives the Na+ translocation (approximate K(m) for Na+ 1 mM), whereas the function of the smallest subunit, delta or GcdD, is unclear. The gene gcdA is part of the 'hydroxyglutarate operon', which does not contain genes coding for the other three subunits. This paper describes that the genes, gcdDCB, are transcribed in this order from a distinct operon. The delta-subunit (GcdD, 12 kDa), with one potential transmembrane helix, probably serves as an anchor for GcdA. The biotin carrier (GcdC, 14 kDa) contains a flexible stretch of 50 amino acid residues (A26-A75), which consists of 34 alanines, 14 prolines, one valine and one lysine. The beta-subunit (GcdB, 39 kDa) comprising 11 putative transmembrane helices shares high amino acid sequence identities with corresponding deduced gene products from Veillonella parvula (80%, clostridial cluster IX), Archaeoglobus fulgidus (61%, Euryarchaeota), Propionigenium modestum (60%, clostridial cluster XIX), Salmonella typhimurium (51%, enterobacteria) and Klebsiella pneumoniae (50%, enterobacteria). Directly upstream of the promoter region of the gcdDCB operon, the 3' end of gctM was detected. It encodes a protein fragment with 73% sequence identity to the C-terminus of the alpha-subunit of methylmalonyl-CoA decarboxylase from V. parvula (MmdA). Hence, it appears that A. fermentans should be able to synthesize this enzyme by expression of gctM together with gdcDCB, but methylmalonyl-CoA decarboxylase activity could not be detected in cell-free extracts. Earlier observations of a second, lower affinity binding site for Na+ of glutaconyl-CoA decarboxylase (apparent K(m) 30 mM) were confirmed by identification of the cysteine residue 243 of GcdB between the putative hellces VII and VIII, which could be specifically protected from alkylation by Na+. The alpha-subunit was purified from an overproducing Escherichia coli strain and was characterized as a putative homotrimer able to catalyse the carboxylation of free biotin.

[Detection of circulating immune complexes in patients with EPH gestosis or intrauterine fetal growth retardation]. / Nachweis von zirkulierenden Immunkomplexen bei Patientinnen mit EPH-Gestose oder intrauteriner fetaler Retardierung.

Sera from 19 patients with EPH gestosis, 22 patients with hypotrophic newborns, and a control group of ten women with normal pregnancy and fetuses were studied for the presence of circulating immune complexes. Both the C1q solid-phase radioimmunoassay and the polyethylene glycol precipitation test revealed significantly higher levels of circulating immune complexes in both groups of patients.