Improved high-performance liquid chromatographic method in the analysis of adenovirus particles

We developed a HPLC method on a novel continuous bed matrix (UNO Q, Bio-Rad) for the direct quantification of adenoviral type 5 (Ad5) particles produced in 293S Human Embryonic Kidney cells and compared this with an existing HPLC method on a conventional ion-exchange resin (Resource Q, Pharmacia). The 293S cell extract contained large amounts of DNA. This contaminated the viral peak on the Resource Q column and only after Benzonase treatment was it possible to quantify the viral particles in the cell extract. In contrast, the virus peak on the UNO Q column was resolved from the DNA which eliminates the need for pretreatment of the sample with Benzonase. Cross-analysis of the Ad5 fraction from the UNO Q column using a size-exclusion HPLC column revealed no additional contaminating peaks. We conclude that the purity of the Ad5 virus peak on the continuous bed matrix UNO Q column was superior to the purity of the virus on the conventional Resource Q column, which is essential for reliable quantification.     

Very small injected samples to study chloroquine and quinine in human serum using capillary-LC and native fluorescence

A comparison between HPLC with conventional fluorescence detection and capillary-LC (microHPLC) with native laser-induced fluorescence (LIF) detection was done to determine chloroquine (CQ) and quinine (Q) in human serum. HPLC experiments were run with parameters of the conventional fluorimeter set at the highest level of sensitivity. Results were compared with those obtained on microHPLC coupled to a ZETALIF (He-Cd 325 nm) detector which provided a 50-fold increase in sensitivity. In microHPLC-LIF injection volumes were 200 nL instead of 10 microL in conventional HPLC. The separation was completed within 3 min (6 min on HPLC). The limit of detection on microHPLC-LIF was 1.9 and 1.3 fmol for CQ and Q, respectively. Both experiments were validated on serum samples. The mean recovery was more than 95% for CQ and Q. The intra- and inter-day precision and accuracy were found to be within the acceptable limits (<10%).     

Coenzyme Q10 in human blood: Native levels and determinants of oxidation during processing and storage

Coenzyme Q10 (Q10) is present in the circulation mainly in its reduced form (ubiquinol-10; UL10), but oxidizes quickly ex vivo to ubiquinone-10 (UN10). Therefore, native UL10:UN10 ratios, used as markers of redox status and disease risk, are difficult to measure. We established an RP-(U)HPLC method with coulometric detection to measure natively circulating UL10 and UN10 concentrations by adding a ubiquinol/ubiquinone mixture as an internal standard immediately after plasma preparation. This allowed adjustment for unavoidable artificial UL10 oxidation as well as for total losses (or gains) of analytes during sample storage, processing, and analysis because the internal standards exactly paralleled the chemical behavior of Q10. This technique applied to blood (n = 13) revealed Q10 levels of 680–3300 nM with a mean UL10:UN10 ratio of 95:5, which was inversely associated with total Q10 (r = ? 0.69; p = 0.004). The oxidation of UL10 to UN10 was equimolar, increased by O₂, and decreased by lower temperatures or various degassing methods. Although UL10 was stable in blood or when pure in organic solvents at 22 °C, its oxidation was catalyzed dose dependently by α-tocopherol and butylated hydroxytoluene, particularly when present in combination. Key structural features for the catalytic pro-oxidant properties of phenolic antioxidants included two substituents vicinal to the phenolic hydroxyl group.     

Identification of quercetin 3-O-beta-D-glucuronide as an antioxidative metabolite in rat plasma after oral administration of quercetin

The potential beneficial effect of dietary quercetin (3,3',4',5,7-pentahydroxyflavone) has attracted much attention in relation to the prevention of cardiovascular disease. It is generally recognized that dietary quercetin is subject to metabolic conversion resulting in conjugated forms during absorption and circulation. However, no quercetin conjugates have yet been identified from biological fluids or tissues. In the present study, we isolated and characterized two quercetin conjugates from the plasma of quercetin-administered rats. The blood plasma was collected from 26 rats 30 min after oral administration of quercetin (250 mg/kg body weight), concentrated, dissolved in 2% acetic acid aqueous solution (pH 2.65), and extracted with ethyl acetate. Two compounds (P2, P3) were obtained from the extract by repeated reversed-phase HPLC. On the other hand, two quercetin glucuronides were synthesized chemically and identified as quercetin 3-O-beta-D-glucuronide (Q3GA) and quercetin 4'-O-beta-D-glucuronide (Q4'GA), as determined from FABMS, 1H- and 13C-NMR, and HMBC data. The retention times of P2 and P3 in the HPLC chromatogram corresponded to those of Q3GA and Q4'GA, respectively. FABMS data demonstrated that P2 and P3 are quercetin monoglucuronides. 1H-NMR data for P2 were completely in agreement with those for Q3GA. P2 was therefore identified as Q3GA. This is, to our knowledge, the first evidence that Q3GA accumulates in vivo after oral administration of quercetin. Q3GA is likely to act as an effective antioxidant in blood plasma low-density lipoprotein, because this conjugated metabolite was found to possess a substantial antioxidant effect on copper ion-induced oxidation of human plasma low-density lipoprotein as well as 1,1-diphenyl-2-picrylhydrazyl radical-scavenging activity.     

Method development and validation for monitoring in vivo oxidative stress: evaluation of lipid peroxidation and fat-soluble vitamin status by HPLC in rat plasma

Monitoring in vivo oxidative stress implicates the evaluation of damage and defence parameters by well-established, validated methods. We report two optimized and validated HPLC methods for measurement of malondialdehyde (MDA) and fat-soluble vitamins in rat plasma. For the MDA method, optimization experiments of the thiobarbituric acid test resulted in the addition of 1% butylhydroxytoluene to the reaction mixture and in a heating time reduction to 40 min, ensuring inhibition of further lipid peroxidation during the test. Validation experiments showed good linearity, precision and recovery. The use of HPLC with coulometric array detection technology permits simultaneous and sensitive analysis of different fat-soluble vitamins and related compounds (tocopherols, retinoids, carotenoids and coenzyme Q10), which are identified by both retention time and electrochemical characteristics. Furthermore, this method is extended to the analysis of coenzyme Q9, the predominant homologue in rats. Validation experiments with rat plasma gave good results.     

Properties of vagal excitation transfer to the heart atrium]

A mathematical model of pre- and postganglionic parasympathetic nerve fiber excitation transfer is developed. This model gives a measure Q of acetylcholine (ACh) release from presynaptic preganglionic boutons and postganglionic varicosities. When increasing Ca++ the measure Q increases too. Na-ions exert a competitive inhibition. The relationship between Q and the Ca/Na2-quotient is a hyperbolic one. Mn++ inhibits the release of ACh non-competitively. Q increases both by excess potassium and Cs+ depolarization. The ACh release is diminished by Mg++. Ba++ cannot replace the effect of Ca++ on ACh release in Ca++ depleting conditions. Q increases with decreasing pH-level. The ACh release is not significantly influenced by increasing pH, Verapamil (4 mg/l), prostaglandins E2 and F2alpha (20 ng/ml) and substitution of nonpermeable anions for Cl-.     

Biological activity of quercetin-3-<Emphasis Type="Italic">O</Emphasis>-glucoside,a known plant flavonoid

Cytotoxic, phytotoxic, antimicrobial and antioxidant effects of quercetin 3-O-glucoside (Q3G) isolated by HPLC from aerial parts of Prangos ferulaceae was studied by MTT assay, lettuce germination assay, disk diffusion and DPPH method. Our results showed that Q3G exhibits high antioxidant effect with RC₅₀ of 22 μg/mL, it has low cytotoxicity and no antibacterial effects. Q3G exhibits high phytotoxic effect with IC₅₀ value of 282.7 μg/ml, as well. It is assumed that Q3G does not play a defense role in plants and it may act as an allelopatic agent. The article is published in the original.     

Weak periodic signal detection by sine-Wiener-noise-induced resonance in the FitzHugh–Nagumo neuron

Based on the FitzHugh–Nagumo (FHN) neuron model subjected to sine-Wiener (SW) noise, impacts of SW noise on weak periodic signal detection are investigated by calculating response measure Q for characterizing synchronization between the input signal and the output temporal activities of the neuron. It is numerically demonstrated that the response measure Q can achieve the optimal value under appropriate and moderate intensity or correlation time of SW noise, suggesting the occurrence of SW-noise-induced stochastic resonance. Furthermore, the optimal value of Q is sensitive to correlation time. Consequently, the correlation time of SW noise has a great influence on the performance of signal detection in the FHN neuron.     

Direct high-performance liquid chromatography determination of propofol and its metabolite quinol with their glucuronide conjugates and preliminary pharmacokinetics in plasma and urine of man

Propofol (P) is metabolized in humans by oxidation to 1,4-di-isopropylquinol (Q). P and Q are in turn conjugated with glucuronic acid to the respective glucuronides, propofol glucuronide (Pgluc), quinol-1-glucuronide (Q1G) and quinol-4-glucuronide (Q4G). Propofol and quinol with their glucuronide conjugates can be measured directly by gradient high-performance liquid chromatographic analysis without enzymic hydrolysis. The glucuronide conjugates were isolated by preparative HPLC from human urine samples. The glucuronides of P and Q were present in plasma and urine, P and Q were present in plasma, but not in urine. Quinol in plasma was present in the oxidised form, the quinone. Calibration curves of the respective glucuronides were constructed by enzymic deconjugation of isolated samples containing different concentrations of the glucuronides. The limit of quantitation of P and quinone in plasma are respectively 0.119 and 0.138 μg/ml. The limit of quantitation of the glucuronides in plasma are respectively: Pgluc 0.370 μg/ml, Q1G 1.02 μg/ml and Q4G 0.278 μg/ml. The corresponding values in urine are: Pgluc 0.264 μg/ml, Q1G 0.731 μg/ml and Q4G 0.199 μg/ml. A pharmacokinetic profile of P with its metabolites is shown, and some preliminary pharmacokinetic parameters of P and Q glucuronides are given.     

Relationship between hydraulic resistance and leaf morphology in broadleaf Quercus species: a new interpretation of leaf lobation

We investigated the relationship between leaf shape and leaf hydraulic resistance in a set of broadleaf Quercus tree species (Q. cerris, Q. frainetto, Q. petraea, Q. pyrenaica, Q. robur, Q. rubra, Q. velutina). Seedlings of all the studied species were grown under uniform environmental conditions. A new high-pressure flowmeter was designed to measure leaf-blade hydraulic resistance. Leaf shape was characterised by the complexity of leaf outline which was regarded as an estimate of leaf lobation. This was done using the box-counting fractal dimension of the leaf silhouette. Leaf hydraulic resistance was negatively related to leaf lobation. It is suggested that the lower hydraulic resistance in deeply lobed leaves may constitute a mechanism for improving water balance under dry atmospheric conditions.     

Sequential V(A)/Q distributions in the normal rabbit by micropore membrane inlet mass spectrometry.

We developed micropore membrane inlet mass spectrometer (MMIMS) probes to rapidly measure inert-gas partial pressures in small blood samples. The mass spectrometer output was linearly related to inert-gas partial pressure (r(2) of 0.996-1.000) and was nearly independent of large variations in inert-gas solubility in liquid samples. We infused six inert gases into five pentobarbital-anesthetized New Zealand rabbits and used the MMIMS system to measure inert-gas partial pressures in systemic and pulmonary arterial blood and in mixed expired gas samples. The retention and excretion data were transformed into distributions of ventilation-to-perfusion ratios (V(A)/Q) with the use of linear regression techniques. Distributions of V(A)/Q were unimodal and broad, consistent with prior reports in the normal rabbit. Total blood sample volume for each VA/Q distribution was 4 ml, and analysis time was 8 min. MMIMS provides a convenient method to perform the multiple inert-gas elimination technique rapidly and with small blood sample volumes.     

Differential regulation of multiple populations of granules in rat adrenal chromaffin cells by culture duration and cyclic AMP

We employed carbon fiber amperometry to measure the amount of catecholamine released from individual granules (i.e. the quantal size, Q) of rat chromaffin cells. The distribution of Q1/3 of amperometric events could be reasonably described by the summation of at least three Gaussians, suggesting that rat chromaffin cells contained at least three distinct populations of granules, with a small, medium or large modal Q. After 3 days of culture, the mean cellular Q reduced by approximately 14%, which did not arise from a uniform percentage decrease in the Q of every granule. Instead, the rundown involved a > 11% decrease in the proportional release from large Q granules and a > 19% decrease in the modal Q-value of small Q granules. In contrast, when cells were cultured with dibutyryl-cAMP (dBcAMP) for 3 days, their mean cellular Q increased by approximately 38% (relative to time-matched controls). This increase in Q was not associated with any shift in the proportional release from the three populations of granules. Instead, cAMP increased the average amount of catecholamines released from all three populations of granules. Our data raise the possibility that distinct populations of granules in rat chromaffin cells can be regulated either differentially or uniformly.     

Opposite patterns in the annual distribution and time-course of endogenous abscisic acid and indole-3-acetic acid in relation to the periodicity of cambial activity in Eucommia ulmoides Oliv

The seasonal change of free abscisic acid (ABA) and indole-3-acetic acid (IAA) and their relationship with the cambial activity in Eucommia ulmoides trees were investigated by ABA and IAA immunolocalization using primary polyclonal and rhodamine-red fluorescing secondary antibodies, ABA and IAA quantification using high performance liquid chromatography (HPLC), and systematic monitoring of vascular cell layers production. ABA and IAA clearly displayed opposite annual distribution patterns. In the active period (AP), both immunolocalization and HPLC detected an abrupt decrease of ABA, reaching its lowest level in the summer. During dormancy, ABA started increasing in the first quiescence (Q1) (autumn), peaked in the rest (winter), and gradually decreased from the onset of the second quiescence (Q2) (the end of winter). IAA showed a reverse pattern to that of ABA: it sharply increased in AP, but noticeably decreased from the commencement of Q1. Longitudinally, the ABA distribution increased apico-basally, contrasting with IAA. Laterally, most of the ABA was located in mature vascular tissues, whereas the IAA essentially occurred in the cambial region. The concomitant IAA-ABA distribution and seasonal changes in vascular tissues greatly correlated with xylem and phloem cell production, and late wood differentiation and maturation. Interestingly, the application of exogenous ABA to quiescent E. ulmoides branches, in a water-culture system, inhibited external IAA action on cambial activity reactivation. These results suggest that, in E. ulmoides, ABA and IAA might probably interact in the cambial region. The annual cambial activity could be influenced by an IAA:ABA ratio; and ABA might play a key role in vascular cambium dormancy in higher plants. The relationship between hormonal changes and the (particular) annual life cycle of E. ulmoides is also discussed.     

Quantification of coronary microvascular resistance using angiographic images for volumetric blood flow measurement: in vivo validation

Structural coronary microcirculation abnormalities are important prognostic determinants in clinical settings. However, an assessment of microvascular resistance (MR) requires a velocity wire. A first-pass distribution analysis technique to measure volumetric blood flow has been previously validated. The aim of this study was the in vivo validation of the MR measurement technique using first-pass distribution analysis. Twelve anesthetized swine were instrumented with a transit-time ultrasound flow probe on the proximal segment of the left anterior descending coronary artery (LAD). Microspheres were injected into the LAD to create a model of microvascular dysfunction. Adenosine (400 μg·kg(-1)·min(-1)) was used to produce maximum hyperemia. A region of interest in the LAD arterial bed was drawn to generate time-density curves using angiographic images. Volumetric blood flow measurements (Q(a)) were made using a time-density curve and the assumption that blood was momentarily replaced with contrast agent during the injection. Blood flow from the flow probe (Q(p)), coronary pressure (P(a)), and right atrium pressure (P(v)) were continuously recorded. Flow probe-based normalized MR (NMR(p)) and angiography-based normalized MR (NMR(a)) were calculated using Q(p) and Q(a), respectively. In 258 measurements, Q(a) showed a strong correlation with the gold standard Q(p) (Q(a) = 0.90 Q(p) + 6.6 ml/min, r(2) = 0.91, P < 0.0001). NMR(a) correlated linearly with NMR(p) (NMR(a) = 0.90 NMR(p) + 0.02 mmHg·ml(-1)·min(-1), r(2) = 0.91, P < 0.0001). Additionally, the Bland-Altman analysis showed a close agreement between NMR(a) and NMR(p). In conclusion, a technique based on angiographic image data for quantifying NMR was validated using a swine model. This study provides a method to measure NMR without using a velocity wire, which can potentially be used to evaluate microvascular conditions during coronary arteriography.     

Quantification of regional V/Q ratios in humans by use of PET. II. Procedure and normal values

Regional measurements of tissue isotope concentration, made using positron emission tomography (PET), allow tracer models to be used in a quantitative manner to provide topographic distributions of many structural and functional parameters, each derived for the same well-defined lung element. In this paper we describe a technique to measure regional ventilation-perfusion ratios (V/Q), in absolute units, by use of PET and the continuous intravenous infusion of an inert gas isotope, 13N, and report on measurements made in 12 normal subjects (4 smokers). Data were obtained from a single lung section (slice thickness, 1.7 cm full width at half-maximum response to a line source) at the level of the right ventricle in the supine posture during quiet breathing. For the 12 subjects, volume-weighted mean values of V/Q, averaged over individual right and left lung fields, ranged from 0.50 to 1.29. Analysis of these means showed no difference between lungs: right, 0.80 +/- 0.23 SD; left, 0.76 +/- 0.20 SD. Topographically, a systematic fall of V/Q in the ventrodorsal direction was observed in eight of the subjects (mean ventrodorsal difference 0.39, range 0.19-0.90), whereas two showed a clear increase toward dependent lung regions (range 0.16-0.26). Seven of the subjects with a falling ventrodorsal V/Q gradient also exhibited discrete regions of low V/Q at the dorsal lung border. We conclude that, in normal subjects, ventilation and perfusion are generally well matched in the supine posture, but isolated mismatching often occurs in dependent lung regions.     

Quinone mediated ATP production in the filarial parasite Setaria digitata

The cattle filarial parasite Setaria digitata, a facultative anaerobe which is reported to be cyanide insensitive, lacks cytochromes and presents many unique characters. Experiments showed the occurrence of two lower quinones Q6 and Q8 and its rapid synthesis is revealed by a [14C] acetate incorporation study. A schematic quinone mediated hydrogen peroxide production with the generation of ATP through oxidation of substrates has been proposed. Search for specific blockers at the level of quinone might prove to be an effective measure for the control of filarial parasites and thereby filariasis.     

Nonoxidizable ubiquinol derivatives that are suitable for the study of the ubiquinol oxidation site in the cytochrome bc1 complex

Recent X-ray crystallographic analyses of the mitochondrial cytochrome bc1 complex show ubiquinone binding at the Q(i) site, but attempts to show binding of ubiquinol or ubiquinone at the Q(o) site have been unsuccessful, even though the binding of noncompetitive Q(o) site inhibitors near the putative ubiquinol binding pocket is well established. We speculate that ubiquinol binds transiently to the Q(o) site only when both heme b(L) and the iron sulfur cluster are in the oxidized form, an experimental condition difficult to obtain since ubiquinol will be oxidized once bound to the site. Stable binding at the Q(o) site might be achieved by a nonoxidizable ubiquinol-like compound. For this purpose, the isomers 2,3,4-trimethoxy-5-decyl-6-methyl-phenol (TMDMP) and 2,3,4-trimethoxy-5-methyl-6-decyl-phenol (TMMDP) were synthesized from 2,3-dimethoxy-5-methyl-6-decyl-1, 4-benzoquinol (Q0C10) by controlled methylation and separated by TLC and HPLC. The structures of TMDMP and TMMDP were established by 1H-13C-two-dimensional NMR. Both are competitive inhibitors of the cytochrome bc1 complex, with TMDMP being the stronger one. Preliminary results suggest that TMDMP binds tightly enough to make X-ray crystallography of inhibitor-bc1 complex co-crystals feasible. The binding site of TMDMP does not overlap with the binding sites of stigmatellin, MOA-stilbene (MOAS), undecylhydroxydioxobenzothiazole (UHDBT) and myxothaizol.     

Pulmonary arterial endothelial cells affect the redox status of coenzyme Q0

The pulmonary endothelium is capable of reducing certain redox-active compounds as they pass from the systemic venous to the arterial circulation. This may have important consequences with regard to the pulmonary and systemic disposition and biochemistry of these compounds. Because quinones comprise an important class of redox-active compounds with a range of physiological, toxicological, and pharmacological activities, the objective of the present study was to determine the fate of a model quinone, coenzyme Q0 (Q), added to the extracellular medium surrounding pulmonary arterial endothelial cells in culture, with particular attention to the effect of the cells on the redox status of Q in the medium. Spectrophotometry, electron paramagnetic resonance (EPR), and high-performance liquid chromatography (HPLC) demonstrated that, when the oxidized form Q is added to the medium surrounding the cells, it is rapidly converted to its quinol form (QH2) with a small concentration of semiquinone (Q*-) also detectable. The isolation of cell plasma membrane proteins revealed an NADH-Q oxidoreductase located on the outer plasma membrane surface, which apparently participates in the reduction process. In addition, once formed the QH2 undergoes a cyanide-sensitive oxidation by the cells. Thus, the actual rate of Q reduction by the cells is greater than the net QH2 output from the cells.     

Attenuation of blood parameters in smokers and non-smokers after intake of a complex food additive

This report describes an intervention study with healthy volunteers (20 smokers, 28 non-smokers) taking a food additive mainly containing vitamin C (ascorbic acid), vitamin E (alpha-tocopherol), ubiquinone (Q10), vitamin A and zinkoxide for four weeks in a double blind, randomized and placebo controlled manner. Before and after the intervention blood was withdrawn and general blood parameters were analyzed. In addition, lipid soluble antioxidants were analyzed in blood plasma by HPLC and the water soluble antioxidative properties were tested with the enzymic xanthin/xanthinoxidase-reaction. In summary the results show that the smoker-verum group exhibit a significant down regulation of the leukocyte counts. The test for antioxidants show the following significant differences after intervention: Smokers exhibit an increase of both vitamin E and coenzyme Q10 and an attenuation of their (before intervention) clearly increased water soluble-antioxidative potential, non-smokers showed only an increase of vitamin E and trends of an increase of Q10 and water soluble-antioxidative potential. These results may contribute to the discussion of the intrinsic deficiency brought about by smoking and the possible attenuation of part of these deficiency by increasing the intake of certain vitamins or food additives.