Remodeling of photosynthetic electron transport in Synechocystis sp. PCC 6803 for future hydrogen production from water

Photosynthetic microorganisms such as the cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis) can be exploited for the light-driven synthesis of valuable compounds. Thermodynamically, it is most beneficial to branch-off photosynthetic electrons at ferredoxin (Fd), which provides electrons for a variety of fundamental metabolic pathways in the cell, with the ferredoxin-NADP⁺ Oxido-Reductase (FNR, PetH) being the main target. In order to re-direct electrons from Fd to another consumer, the high electron transport rate between Fd and FNR has to be reduced. Based on our previous in vitro experiments, corresponding FNR-mutants at position FNR_K190 (Wiegand, K., et al.: “Rational redesign of the ferredoxin-NADP-oxido-reductase/ferredoxin-interaction for photosynthesis-dependent H₂-production”. Biochim Biophys Acta, 2018) have been generated in Synechocystis cells to study their impact on the cellular metabolism and their potential for a future hydrogen-producing design cell. Out of two promising candidates, mutation FNR_K190D proved to be lethal due to oxidative stress, while FNR_K190A was successfully generated and characterized: The light induced NADPH formation is clearly impaired in this mutant and it shows also major metabolic adaptations like a higher glucose metabolism as evidenced by quantitative mass spectrometric analysis. These results indicate a high potential for the future use of photosynthetic electrons in engineered design cells – for instance for hydrogen production. They also show substantial differences of interacting proteins in an in vitro environment vs. physiological conditions in whole cells.     

Photosynthesis and photorespiration in a mutant of the cyanobacterium Synechocystis PCC 6803 lacking carboxysomes

A mutant of the cyanobacterium Synechocystis PCC 6803 was obtained by replacing the gene of the carboxylation enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) with that of the photosynthetic bacterium Rhodospirillum rubrum. This mutant consequently lacks carboxysomes — the protein complexes in which the original enzyme is packed. It is incapable of growing at atmospheric CO₂ levels and has an apparent photosynthetic affinity for inorganic carbon (C_i) which is 1000 times lower than that of the wild type, yet it accumulates more C_i than the wild type. The mutant appears to be defective in its ability to utilize the intracellular C_i pool for photosynthesis. Unlike the carboxysomal carboxylase activity of Rubisco, which is almost insensitive to inhibition by O₂ in vitro, the soluble enzyme is competitively inhibited by O₂. The photosynthetic rate and C_i compensation point of the wild type were hardly affected by low O₂ levels. Above 100 μM O₂, however, both parameters became inhibited. The CO₂ compensation point of the mutant was linearly dependent on O₂ concentration. The higher sensitivity of the mutant to O₂ inhibition than that expected from in-vitro kinetics parameters of Rubisco, indicates a low capacity to recycle photorespiratory metabolites to Calvin-cycle intermediates.     

Determination of midazolam and its metabolites in serum microsamples by high-performance liquid chromatography and its application to pharmacokinetics in rats

A single-solvent extraction step high-performance liquid chromatographic method is described for quantitating midazolam and its two hydroxy metabolites in rat serum microsamples (50 μl). The separation used a 2 mm I.D. reversed-phase Symmetry C₁₈ column with an isocratic mobile phase consisting of methanol-acetonitrile-14.9 mM sodium acetate in water at pH 3.0 (10:23:67, v/v). The detection limit was 10 ng/ml for all the compounds using an ultraviolet detector operated at 230 nm. The method was used to study the pharmacokinetics of midazolam after an intravenous bolus dose (0.75 mg/kg).     

Temperature-Induced Changes in the Fatty Acid Composition of the Cyanobacterium, Synechocystis PCC6803

Changes in glycerolipid and fatty acid composition with a change in growth temperature were studied in the cyanobacterium, Synechocystis PCC6803. Under isothermal growth conditions, temperature did not significantly affect the composition of the various classes of lipids, but a decrease in temperature altered the degree of unsaturation of C₁₈ acids at the sn-1 position, but not that of C₁₆ acids at the sn-2 position of the glycerol moiety in each class of lipids. When the growth temperature was shifted from 38°C to 22°C, the desaturation of C₁₈ acids, but not that of C₁₆ acids, was stimulated. The desaturation of fatty acids occurred only in the light and was inhibited by chloramphenicol, rifampicin and 3-(3,4-dichlorophenyl)-1, 1-dimethylurea, but not by cerulenin, an inhibitor for fatty acid synthesis. These findings suggest that desaturase activities are induced after a shift from a higher to a lower temperature, and that the desaturation of fatty acids is connected with the reactions involved in photosynthetic electron transport.     

Simultaneous determination of ivabradine and its metabolites in human plasma by liquid chromatography–tandem mass spectrometry

A rapid, selective, sensitive and reproducible liquid chromatographic method with tandem mass spectrometric detection has been developed and validated for the analysis of a new specific bradycardic agent, ivabradine (S 16257) and six potentially active metabolites in human plasma. Isolation of these compounds and of the internal standard was performed by an automated solid-phase extraction system using Oasis cartridges. Separation and detection of ivabradine and its metabolites were achieved using a C₁₈ column and a MS–MS detector with a positive electrospray ionization source. Ivabradine and its metabolites gave a linear response ranging from 0.1 or 0.2 to 20 ng/ml and the limits of quantitation ranged from 0.1 to 0.2 ng/ml using a 0.5 ml plasma sample size. A complete validation demonstrated the method to be accurate, precise and specific for the simultaneous quantification of ivabradine and its metabolites in human plasma. The method was subsequently applied to the quantitative determination of ivabradine and its metabolites in human plasma samples from healthy volunteers participating in a clinical study to provide pharmacokinetic data.     

Reversed-phase high-performance liquid chromatography analysis of 6-thioguanine applicable to pharmacologic studies in humans

6-Thioguanine (6TG) and its metabolites were analyzed in human plasma with a reversed-phase high-performance liquid chromatographic method. 6TG and related compounds were extracted from plasma with an equal volume of 2 N perchloric acid at a 50–100% recovery efficiency. The neutralized extracts were chromatographed on a μBondapak C₁₈ column by two separate isocratic conditions. 6TG, 6-thiouric acid, 6-thioxanthine, 6-thioguanosine, and 6-methylthiouric acid were analyzed with 0.01 M sodium acetate, pH 3.5–10% methanol as the mobile phase and 340 nm for detection. 6-Methylthioguanine and three unknown metabolites were separated with acetate—25% methanol and 310 nm detection. One of the unknowns was identified as 6-methylthioguanosine. External standard calibration was used for quantitation. The 6TG detection limit was 0.8 nmol/ml in plasma.     

Simultaneous determination of the peptide-mitomycin KW-2149 and its metabolites in plasma by high-performance liquid chromatography

A gradient high-performance liquid chromatographic (HPLC) method is described for the quantification of KW-2149 and its two major metabolites in plasma. The method involves a sample clean-up by solid-phase extraction on C₁₈ columns, separation of the respective compounds by HPLC on a YMC ODS-AQ column (5-μm particle size, 150×6 mm I.D.), using a methanol–water gradient system as an eluent, and measurement by UV absorbance detection at 375 nm. The limits of quantitation were 10 ng/ml for KW-2149 and M-16, and 15 ng/ml for M-18. Recoveries from plasma were higher than 92% on C₁₈ extraction columns. Intra-day precision, expressed as %C.V., was between 1.4 and 6.5%. Intra-day accuracy ranged from 94 to 107%. Precision and accuracy of variability of inter-assays increased somewhat; however, were still within acceptable ranges. The ability of the method to quantify KW-2149 and two major metabolites simultaneously, with precision, accuracy and sensitivity, make it useful in monitoring the fate of this new mitomycin in cancer patients.     

Quantitative high-performance liquid chromatographic determination of acrolein in plasma after derivatization with Luminarin 3

A rapid, sensitive and specific high-performance liquid chromatographic method for the quantification of acrolein (1), one of the toxic metabolites of oxazaphosphorine alkylating agents (cyclophosphamide and ifosfamide) was developed. Condensation of acrolein with Luminarin^® 3 afforded a fluorescent derivative that could be specifically detected and quantified. Chromatographic conditions involved a C₁₈ RP column Uptisphere and a gradient elution system to optimize resolution and time analysis. The method showed high sensitivity with a limit of detection of 100 pmol/ml and a limit of quantification of 300 pmol/ml. This technique is particularly suitable for pharmacokinetic studies on plasma of oxazaphosphorine-receiving patients.     

Determination of N-acetylation phenotype using caffeine as a metabolic probe and high-performance liquid chromatography with either ultraviolet detection or electrospray mass spectrometry

A rapid, sensitive method using liquid chromatography–electrospray mass spectrometry (LC–ES-MS) was developed and evaluated for the simultaneous quantitative determination of caffeine metabolites 1U, 1X and AAMU in human urine. This method involved a simple dilution of urine samples. The chromatographic separation was achieved on a C₁₈ reversed-phase column using a gradient of acetonitrile in 2 mM, pH 3.0 ammonium formate as mobile phase. After ionisation in an electrospray source, mass spectrometric detection was performed in the negative ion, selected ion monitoring mode. This method yielded acceptable accuracy and precision within the range 0.25–50 μg/ml. This analytical method was applied to investigate the N-acetylator phenotype of HIV-infected patients and compared with high-performance liquid chromatography with UV detection. Its specificity was better, which appeared to be absolutely necessary to prevent errors in metabolic ratios and phenotype interpretation.     

Remodeling of phosphatidylglycerol in Synechocystis PCC6803

The phosphatidylglycerol deficient ΔpgsA mutant of Synechocystis PCC6803 provided a unique experimental system for investigating in vivo retailoring of exogenously added dioleoylphosphatidylglycerol in phosphatidylglycerol-depleted cells. Gas chromatographic analysis of fatty acid composition suggested that diacyl-phosphatidylglycerols were synthesized from the artificial synthetic precursor. The formation of new, retailored lipid species was confirmed by negative-ion electrospray ionization–Fourier-transform ion cyclotron resonance and ion trap tandem mass spectrometry. Various isomeric diacyl-phosphatidylglycerols were identified indicating transesterification of the exogenously added dioleoylphosphatidyl-glycerol at the sn-1 or sn-2 positions. Polyunsaturated fatty acids were incorporated selectively into the sn-1 position. Our experiments with Synechocystis PCC6803/ΔpgsA mutant cells demonstrated lipid remodeling in a prokaryotic photosynthetic bacterium. Our data suggest that the remodeling of diacylphosphatidylglycerol likely involves reactions catalyzed by phospholipase A₁ and A₂ or acyl-hydrolase, lysophosphatidylglycerol acyltransferase and acyl-lipid desaturases.     

Assay of the antiangiogenic compound TNP-470, and one of its metabolites, AGM-1883, by reversed-phase high-performance liquid chromatography in plasma

This paper describes a reversed-phase, high-performance liquid chromatographic (HPLC) method for the isolation, detection, and quantification of TNP-470 (I) and one of its active metabolites, AGM-1883 (II), from plasma. These compounds are initially extracted from plasma with an organic solvent and then separated from one another on a C₁₈ column. Those fractions eluting from the C₁₈ column and containing either I or II are then derivatized through their epoxide moieties with sodium 8-quinolinethiolate (SQT). This derivatization produces fluorescent species that are isolated and quantified by a second reversed-phase HPLC analysis. The assay yields a lower limit of reliable quantification of 2.5 ng/ml and is linear to a concentration at least as high as 160 ng/ml. The inter-assay percent coefficient of variation is less than 18%.     

Determination of lumefantrine in rat plasma by liquid–liquid extraction using LC–MS/MS with electrospray ionization: Assay development,validation and application to a pharmacokinetic study

A simple, sensitive and rapid method for the analysis of lumefantrine in rat plasma using liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) was developed. Detection was performed by positive ion electrospray ionization (ESI) in multiple reaction monitoring (MRM) mode. The method included a chromatographic run of 5 min using a C₁₈ analytical column and the calibration curve was linear over the concentration range of 2–500 ng/mL with a correlation coefficient (r) of 0.996 or better. The intra- and inter-day assay precision ranged from 1.5 to 7.5% and 5.5 to 7.7%, respectively, and intra- and inter-day assay accuracy was between 91.3–109.7% and 97.0–104.7%, respectively. The method was successfully applied for the pharmacokinetic study in rats.     

Biological Effects of Trichoderma harzianum Peptaibols on Mammalian Cells

Trichoderma species isolated from water-damaged buildings were screened for toxicity by using boar sperm cells as indicator cells. The crude methanolic cell extract from Trichoderma harzianum strain ES39 inhibited the boar sperm cell motility at a low exposure concentration (50% effective concentration, 1 to 5 μg [dry weight] ml of extended boar semen⁻¹). The same exposure concentration depleted the boar sperm cells of NADH₂. Inspection of the exposed boar sperm cells by transmission electron microscopy revealed damage to the plasma membrane. By using the black lipid membrane technique, it was shown that the semipurified metabolites (eluted from a SepPak C₁₈ cartridge) of T. harzianum strain ES39 induced voltage-dependent conductivity. The high-performance liquid chromatography-purified metabolites of T. harzianum strain ES39 dissipated the mitochondrial membrane potential (Δψ_m) of human lung epithelial carcinoma cells (cell line A549). The semipurified metabolites (eluted from a SepPak C₁₈ cartridge) of T. harzianum strain ES39 were analyzed by mass spectrometry (MS). Matrix-assisted laser desorption ionization and nanoflow electrospray ionization MS revealed five major peptaibols, each of which contained 18 residues and had a mass ranging from 1,719 to 1,775 Da. Their partial amino acid sequences were determined by collision-induced dissociation tandem MS.     

Detection and separation of the anthrapyrazole CI-941 and its metabolites in serum and urine by high-performance liquid chromatography

A high-performance liquid chromatographic method using ion-pairing chromatography on reversed-phase C₁₈ material with a mobile phase of acetonitrile—water (19:81, v/v) containing 5 mM 1-pentanesulfonic acid was developed for the detection and separation of the anthrapyrazole CI-941 (I) and its metabolites. After sample clean-up with solid-phase extraction, I and its metabolites were measurable at a wavelength of 491 nm. A detection limit of 5 ng/ml was achievable for I. The dicarboxylic acid derivative and the isomers of the monocarboxylic acid derivative could be separated. Application of the method to a human pharmacokinetic study showed two and four metabolites of I in serum and urine respectively.     

Simultaneous determination of nerisopam,a novel anxiolytic agent showing polymorphic metabolism,and its N-acetyl metabolite from human plasma by a validated high-performance liquid chromatographic method

A sensitive reversed-phase high-performance liquid chromatographic method with ultraviolet absorbance detection has been developed to simultaneously determine the concentrations of nerisopam (EGIS-6775) and its N-acetyl metabolite (EGIS-7649) from human plasma. The separation of the investigated compounds and internal standard was achieved on a Nucleosil 7 C₁₈ column with 2 mM heptanesulphonic acid containing 0.04 M phosphoric acid-acetonitrile-methanol (70:25:5 v/v), pH 2.7 mobile phase. The detection was performed at 385 nm. The compounds were isolated from plasma by Bakerbond C₁₈ solid-phase extraction. The limit of quantitation was 10 ng/ml plasma for each compound investigated. The assay has been validated with respect to accuracy, precision and system suitability. All validated parameters were found to be within the necessary limits. On the basis of the sensitivity, linearity and validation parameters, the developed analytical method was found to be suitable for the determination of nerisopam and its N-acetyl metabolite from human plasma and for application in pharmacokinetic studies and human drug monitoring. The pharmacokinetic parameters obtained from twelve human volunteers are reported. It was found that nerisopam acetylation is polymorphic: the volunteers with fast or slow acetylator phenotypes produced significantly different plasma concentrations. In slow acetylator phenotypes the concentration of nerisopam was considerably higher in plasma, while the level of its acetyl metabolite was higher in plasma of fast acetylators.     

Simultaneous determination of phenylbutazone and its metabolites in plasma and urine by high-performance liquid chromatography

A high-performance liquid chromatographic method was developed for the simultaneous determination of phenylbutazone and its metabolites, oxyphenbutazone and γ-hydroxyphenylbutazone, in plasma and urine. Samples were acidified with hydrochloric acid and extracted with benzene—cyclohexane (1:1, v/v). The extract was redissolved in methanol and chromatographed on a μBondapak C₁₅ column using a mobile phase of methanol—0.01 M sodium acetate buffer (pH 4.0) in a linear gradient (50 to 100% methanol at 5%/min; flow-rate 2.0 ml/min) in a high-performance liquid chromatograph equipped with an ultra-violet absorbance detector (254 nm). The detection limit for phenylbutazone, oxyphenbutazone and for γ-hydroxyphenylbutazone was 0.05 μg/ml.A precise and sensitive assay for the determination of phenylbutazone and its metabolites was established.     

Simultaneous determination of m-nisoldipine and its three metabolites in rat plasma by liquid chromatography–mass spectrometry

A simple, sensitive and selective liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the simultaneous determination of m-nisoldipine and its three metabolites in rat plasma has been developed using nitrendipine as an internal standard (IS). Following liquid–liquid extraction, the analytes were separated using an isocratic mobile phase on a reverse phase C₁₈ column and analyzed by MS in the multiple reaction monitoring (MRM) mode. To avoid contamination by residual sample in the injection syringe, a special injection protocol was developed. We found that m-nisoldipine, metabolite M1 and IS could be ionized under positive or negative electrospray ionization conditions, whereas metabolite M and M2 could only be ionized in the positive mode. The mass spectrometry fragmentation pathways for these analytes are analyzed and discussed herein. The total analysis time required less than 5 min per sample. We employed this method successfully to study the metabolism of m-nisoldipine when it was orally administered to rats at a dose of 9 mg/kg. Three metabolites of m-nisoldipine and an unknown compound of molecular weight 386 were found for the first time in rat plasma. The concentration of the potentially active metabolite was approximately equal to its parent compound concentration.     

Ultrasensitive determination of NE-100, a novel sigma ligand,in human plasma by liquid chromatography and electrospray ionization tandem mass spectrometry combined with a column-switching technique

For the highly sensitive and selective determination of NE-100, a novel sigma ligand, at levels of low picogram per milliliter of human plasma, a method with excellent reliability employing liquid chromatography (LC)–electrospray ionization (ESI) tandem mass spectrometry (MS–MS) combined with a column-switching technique has been developed. The method involves the use of a stable isotope labeled compound as the internal standard (I.S.), liquid–solid extraction of a plasma specimen with a C₈ cartridge, automated on-line clean-up on a short trapping column, subsequent separation on a micro-bore C₁₈ column and detection with ESI-MS–MS using m/z 356 ([M+H]⁺) as a precursor ion and m/z 105 as a product ion in a selected reaction monitoring mode. The detection and the quantification limits of NE-100 in plasma were 0.5 pg/ml with a signal-to-noise ratio (S/N) of 3 and 2.3 pg/ml, respectively, with an S/N of 21. The good linearity of the calibration graph was obtained in the range of 2.3∼907.0 pg/ml with excellent reliability. The developed method was applied to the determination of NE-100 in plasma obtained from the clinical trail.     

Determination of urinary 18β-glycyrrhetinic acid by gas chromatography and its clinical application in man

A sensitive and quantitative gas chromatographic assay for the determination of 18β-glycyrrhetinic acid (18β-GA), the main metabolite of glycyrrhizin after oral licorice consumption in human urine, has been developed and validated. For the extraction of 18β-GA from urine two Sep-Pak C₁₈ extractions, hydrolysis with Helix pomatia and three liquid–liquid extractions were performed, using 18α-glycyrrhetinic acid (18α-GA) as internal standard. Both 18β-GA and internal standard were converted into their pentafluorobenzyl-ester/trimethylsilyl-ether derivatives and detected by flame ionization detection using a WCOT-fused-silica capillary column. Good quality control data were obtained in precision and accuracy tests. The detection limit of the gas chromatographic method was 10 μg/l with a urine volume of 10 ml. A detection limit of 3 μg/l was obtained by performing GC–MS. The GC method was used to monitor the urinary excretion of 18β-GA after licorice consumption by two healthy volunteers and a patient suspected of licorice abuse. Furthermore, it was shown that this GC assay enables to detect other metabolites related to licorice consumption.     

An LC/MS/MS method for stable isotope dilution studies of β-carotene bioavailability,bioconversion, and vitamin A status in humans

Isotope dilution is currently the most accurate technique in humans to determine vitamin A status and bioavailability/bioconversion of provitamin A carotenoids such as β-carotene. However, limits of MS detection, coupled with extensive isolation procedures, have hindered investigations of physiologically-relevant doses of stable isotopes in large intervention trials. Here, a sensitive liquid chromatography-tandem mass spectrometry (LC/MS/MS) analytical method was developed to study the plasma response from coadministered oral doses of 2 mg [¹³C₁₀]β-carotene and 1 mg [¹³C₁₀]retinyl acetate in human subjects over a 2 week period. A reverse phase C₁₈ column and binary mobile phase solvent system separated β-carotene, retinol, retinyl acetate, retinyl linoleate, retinyl palmitate/retinyl oleate, and retinyl stearate within a 7 min run time. Selected reaction monitoring of analytes was performed under atmospheric pressure chemical ionization in positive mode at m/z 537→321 and m/z 269→93 for respective [¹²C]β-carotene and [¹²C] retinoids; m/z 547→330 and m/z 274→98 for [¹³C₁₀]β-carotene and [¹³C₅] cleavage products; and m/z 279→100 for metabolites of [¹³C₁₀]retinyl acetate. A single one-phase solvent extraction, with no saponification or purification steps, left retinyl esters intact for determination of intestinally-derived retinol in chylomicrons versus retinol from the liver bound to retinol binding protein. Coadministration of [¹³C₁₀]retinyl acetate with [¹³C₁₀]β-carotene not only acts as a reference dose for inter-individual variations in absorption and chylomicron clearance rates, but also allows for simultaneous determination of an individual''s vitamin A status.