The Effects of Lung Protective Ventilation or Hypercapnic Acidosis on Gas Exchange and Lung Injury in Surfactant Deficient Rabbits

Background

Permissive hypercapnia has been shown to reduce lung injury in subjects with surfactant deficiency. Experimental studies suggest that hypercapnic acidosis by itself rather than decreased tidal volume may be a key protective factor.

Objectives

To study the differential effects of a lung protective ventilatory strategy or hypercapnic acidosis on gas exchange, hemodynamics and lung injury in an animal model of surfactant deficiency.

Methods

30 anesthetized, surfactant-depleted rabbits were mechanically ventilated (FiO₂ = 0.8, PEEP = 7cmH₂O) and randomized into three groups: Normoventilation-Normocapnia (NN)-group: tidal volume (Vt) = 7.5 ml/kg, target PaCO₂ = 40 mmHg; Normoventilation-Hypercapnia (NH)-group: Vt = 7.5 ml/kg, target PaCO₂ = 80 mmHg by increasing FiCO₂; and a Hypoventilation-Hypercapnia (HH)-group: Vt = 4.5 ml/kg, target PaCO₂ = 80 mmHg. Plasma lactate and interleukin (IL)-8 were measured every 2 h. Animals were sacrificed after 6 h to perform bronchoalveolar lavage (BAL), to measure lung wet-to-dry weight, lung tissue IL-8, and to obtain lung histology.

Results

PaO₂ was significantly higher in the HH-group compared to the NN-group (p<0.05), with values of the NH-group between the HH- and NN-groups. Other markers of lung injury (wet-dry-weight, BAL-Protein, histology-score, plasma-IL-8 and lung tissue IL-8) resulted in significantly lower values for the HH-group compared to the NN-group and trends for the NH-group towards lower values compared to the NN-group. Lactate was significantly lower in both hypercapnia groups compared to the NN-group.

Conclusion

Whereas hypercapnic acidosis may have some beneficial effects, a significant effect on lung injury and systemic inflammatory response is dependent upon a lower tidal volume rather than resultant arterial CO₂ tensions and pH alone.     

Streptococcal dTDP‐L‐rhamnose biosynthesis enzymes: functional characterization and lead compound identification

Biosynthesis of the nucleotide sugar precursor dTDP‐L‐rhamnose is critical for the viability and virulence of many human pathogenic bacteria, including Streptococcus pyogenes (Group A Streptococcus; GAS), Streptococcus mutans and Mycobacterium tuberculosis. Streptococcal pathogens require dTDP‐L‐rhamnose for the production of structurally similar rhamnose polysaccharides in their cell wall. Via heterologous expression in S. mutans, we confirmed that GAS RmlB and RmlC are critical for dTDP‐L‐rhamnose biosynthesis through their action as dTDP‐glucose‐4,6‐dehydratase and dTDP‐4‐keto‐6‐deoxyglucose‐3,5‐epimerase enzymes respectively. Complementation with GAS RmlB and RmlC containing specific point mutations corroborated the conservation of previous identified catalytic residues. Bio‐layer interferometry was used to identify and confirm inhibitory lead compounds that bind to GAS dTDP‐rhamnose biosynthesis enzymes RmlB, RmlC and GacA. One of the identified compounds, Ri03, inhibited growth of GAS, other rhamnose‐dependent streptococcal pathogens as well as M. tuberculosis with an IC₅₀ of 120–410 µM. Importantly, we confirmed that Ri03 inhibited dTDP‐L‐rhamnose formation in a concentration‐dependent manner through a biochemical assay with recombinant rhamnose biosynthesis enzymes. We therefore conclude that inhibitors of dTDP‐L‐rhamnose biosynthesis, such as Ri03, affect streptococcal and mycobacterial viability and can serve as lead compounds for the development of a new class of antibiotics that targets dTDP‐rhamnose biosynthesis in pathogenic bacteria.     

1064 nm Nd:YAG laser light affects transmembrane mitochondria respiratory chain complexes

Photobiomodulation (PBM) is a non‐plant‐cell manipulation through a transfer of energy by means of light sources at the non‐ablative or thermal intensity. Authors showed that cytochrome‐c‐oxidase (complex IV) is the specific chromophore's target of PBM at the red (600‐700 nm) and NIR (760‐900 nm) wavelength regions. Recently, it was suggested that the infrared region of the spectrum could influence other chromospheres, despite the interaction by wavelengths higher than 900 nm with mitochondrial chromophores was not clearly demonstrated. We characterized the interaction between mitochondria respiratory chain, malate dehydrogenase, a key enzyme of Krebs cycle, and 3‐hydroxyacyl‐CoA dehydrogenase, an enzyme involved in the β‐oxidation (two mitochondrial matrix enzymes) with the 1064 nm Nd:YAG (100mps and 10 Hz frequency mode) irradiated at the average power density of 0.50, 0.75, 1.00, 1.25 and 1.50 W/cm² to generate the respective fluences of 30, 45, 60, 75 and 90 J/cm². Our results show the effect of laser light on the transmembrane mitochondrial complexes I, III, IV and V (adenosine triphosphate synthase) (window effects), but not on the extrinsic mitochondrial membrane complex II and mitochondria matrix enzymes. The effect is not due to macroscopical thermal change. An interaction of this wavelength with the Fe‐S proteins and Cu‐centers of respiratory complexes and with the water molecules could be supposed.      

Functional and structural characterization of a prokaryotic peptide transporter with features similar to mammalian PEPT1

The ydgR gene of Escherichia coli encodes a protein of the proton-dependent oligopeptide transporter (POT) family. We cloned YdgR and overexpressed the His-tagged fusion protein in E. coli BL21 cells. Bacterial growth inhibition in the presence of the toxic phosphonopeptide alafosfalin established YgdR functionality. Transport was abolished in the presence of the proton ionophore carbonyl cyanide p-chlorophenylhydrazone, suggesting a proton-coupled transport mechanism. YdgR transports selectively only di- and tripeptides and structurally related peptidomimetics (such as aminocephalosporins) with a substrate recognition pattern almost identical to the mammalian peptide transporter PEPT1. The YdgR protein was purified to homogeneity from E. coli membranes. Blue native-polyacrylamide gel electrophoresis and transmission electron microscopy of detergent-solubilized YdgR suggest that it exists in monomeric form. Transmission electron microscopy revealed a crown-like structure with a diameter of approximately 8 nm and a central density. These are the first structural data obtained from a proton-dependent peptide transporter, and the YgdR protein seems an excellent model for studies on substrate and inhibitor interactions as well as on the molecular architecture of cell membrane peptide transporters.     

Accurate quantification of dimethylamine (DMA) in human urine by gas chromatography-mass spectrometry as pentafluorobenzamide derivative: evaluation of the relationship between DMA and its precursor asymmetric dimethylarginine (ADMA) in health and disease

Dimethylamine [DMA, (CH(3))(2)NH)] is abundantly present in human urine. Main sources of urinary DMA have been reported to include trimethylamine N-oxide, a common food component, and asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide (NO) synthesis. ADMA is excreted in the urine in part unmetabolized and in part after hydrolysis to DMA by dimethylarginine dimethylaminohydrolase (DDAH). Here we describe a GC-MS method for the accurate and rapid quantification of DMA in human urine. The method involves use of (CD(3))(2)NH as internal standard, simultaneous derivatization with pentafluorobenzoyl chloride and extraction in toluene, and selected-ion monitoring of m/z 239 for DMA and m/z 245 for (CD(3))(2)NH in the electron ionization mode. GC-MS analysis of urine samples from 10 healthy volunteers revealed a DMA concentration of 264+/-173 microM equivalent to 10.1+/-1.64 micromol/mmol creatinine. GC-tandem MS analysis of the same urine samples revealed an ADMA concentration of 27.3+/-15.3 microM corresponding to 1.35+/-1.2 micromol/mmol creatinine. In these volunteers, a positive correlation (R=0.83919, P=0.0024) was found between urinary DMA and ADMA, with the DMA/ADMA molar ratio being 10.8+/-6.2. Elevated excretion rates of DMA (52.9+/-18.5 micromol/mmol creatinine) and ADMA (3.85+/-1.65 micromol/mmol creatinine) were found by the method in 49 patients suffering from coronary artery disease, with the DMA/ADMA molar ratio also being elevated (16.8+/-12.8). In 12 patients suffering from end-stage liver disease, excretion rates of DMA (47.8+/-19.7 micromol/mmol creatinine) and ADMA (5.6+/-1.5 micromol/mmol creatinine) were found to be elevated, with the DMA/ADMA molar ratio (9.17+/-4.2) being insignificantly lower (P=0.46). Between urinary DMA and ADMA there was a positive correlation (R=0.6655, P<0.0001) in coronary artery disease, but no correlation (R=0.27339) was found in end-stage liver disease.     

Accurate quantification of dimethylamine (DMA) in human plasma and serum by GC-MS and GC-tandem MS as pentafluorobenzamide derivative in the positive-ion chemical ionization mode

Dimethylamine (DMA) circulates in human blood and is excreted in the urine. Major precursor for endogenous DMA is asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide (NO) synthesis. ADMA is hydrolyzed to DMA and L-citrulline by dimethylarginine dimethylaminohydrolase (DDAH). In previous work, we reported a GC-MS method for the quantification of DMA in human urine. This method involves simultaneous derivatization of endogenous DMA and the internal standard (CD(3))(2)NH by pentafluorobenzoyl chloride (PFBoylCl) and extraction of the pentafluorobenzamide derivatives by toluene. In the present work, we optimized this derivatization/extraction procedure for the quantitative determination of DMA in human plasma. Optimized experimental parameters included vortex time and concentration of PFBoylCl, carbonate and internal standard. The GC-MS method was thoroughly validated and applied to measure DMA concentrations in human plasma and serum samples. GC-MS quantification was performed by selected-ion monitoring of the protonated molecules at m/z 240 for DMA and m/z 246 for (CD(3))(2)NH in the positive-ion chemical ionization mode. Circulating DMA concentration in healthy young women (n=18) was determined to be 1.43+/-0.23 micaroM in serum, 1.73+/-0.17 microM in lithium heparin plasma, and 9.84+/-1.43 microM in EDTA plasma. DMA was identified as an abundant contaminant in EDTA vacutainer tubes (9.3+/-1.9 nmol/monovette, n=6). Serum and lithium heparin vacutainer tubes contained considerably smaller amounts of DMA (0.42+/-0.01 and 0.95+/-0.01 nmol/monovette, respectively, each n=6). Serum is recommended as the most appropriate matrix for measuring DMA in human blood. The present GC-MS method should be useful for the determination of systemic and whole body DDAH activity by measuring circulating and excretory DMA in experimental and clinical studies.     

Identification of Protein-Tyrosine Phosphatases in Archaea

Protein-tyrosine dephosphorylation is a major mechanism in cellular regulation. A large number of protein-tyrosine phosphatases is known from Eukarya, and more recently bacterial homologues have also been identified. By employing conserved sequence patterns from both eukaryotic and bacterial protein-tyrosine phosphatases, we have identified three homologous sequences in two of the four complete archaeal genomes. Two hypothetical open reading frames in the genome of Methanococcus jannaschii (MJ0215 and MJECL20) and one in the genome of Pyrococcus horikoshii (PH1732) clearly bear all the conserved residues of this family. No homologues were found in the genomes of Archaeoglobus fulgidus and Methanobacterium thermoautotrophicum. This is the first report of protein-tyrosine phosphatase sequences in Archaea. Received: 29 April 1998 / Accepted: 27 November 1998     

Gas chromatographic-tandem mass spectrometric quantification of free 3-nitrotyrosine in human plasma at the basal state

A fully validated gas chromatographic-tandem mass spectrometric (GC-tandem MS) method for the accurate and precise quantification of free 3-nitrotyrosine in human plasma at the basal state is described. In the plasma of 11 healthy humans a mean concentration of 2.8 nM (range 1.4-4.2 nM) for free 3-nitrotyrosine was determined by this method. This is the lowest concentration reported for free 3-nitrotyrosine in plasma of healthy humans. The presence of endogenous free 3-nitrotyrosine in human plasma was unequivocally shown by generating a daughter mass spectrum. Various precautions had to be taken to avoid artifactual formation of 3-nitrotyrosine from nitrate during sample treatment. Endogenous plasma 3-nitrotyrosine and 3-nitro-l-[(2)H(3)]tyrosine added for use as internal standard were isolated by high-performance liquid chromatographic (HPLC) analysis of 200-microl aliquots of plasma ultrafiltrate samples (20 kDa cut-off), extracted from a single HPLC fraction by solid-phase extraction, derivatized to their n-propyl ester-pentafluoropropionyl amide-trimethylsilyl ether derivatives, and quantified by GC-tandem MS. Overall recovery was determined as 50 +/- 5% using 3-nitro-l-[(14)C(9)]tyrosine. The limit of detection of the method was 4 amol of 3-nitrotyrosine, while the limit of quantitation was 125 pM using 3-nitro-l-[(14)C(9)]tyrosine. 3-Nitrotyrosine added to human plasma at 1 nM was quantitated with an accuracy of > or = 80% and a precision of > or = 94%. The method should be useful to investigate the utility of plasma free 3-nitrotyrosine as an indicator of nitric oxide ((.)NO)-associated oxidative stress in vivo in humans.