Absolute quantification of resting oxygen metabolism and metabolic reactivity during functional activation using QUO2 MRI.

We have recently described an extension of calibrated MRI, which we term QUO2 (for QUantitative O(2) imaging), providing absolute quantification of resting oxidative metabolism (CMRO(2)) and oxygen extraction fraction (OEF(0)). By combining BOLD, arterial spin labeling (ASL) and end-tidal O(2) measurements in response to hypercapnia, hyperoxia and combined hyperoxia/hypercapnia manipulations, and the same MRI measurements during a task, a comprehensive set of vascular and metabolic measurements can be obtained using a generalized calibration model (GCM). These include the baseline absolute CBF in units of ml/100g/min, cerebrovascular reactivity (CVR) in units of %Δ CBF/mm Hg, M in units of percent, OEF(0) and CMRO(2) at rest in units of µmol/100g/min, percent evoked CMRO(2) during the task and n, the value for flow-metabolic coupling associated with the task. The M parameter is a calibration constant corresponding to the maximal BOLD signal that would occur upon removal of all deoxyhemoglobin. We have previously shown that the GCM provides estimates of the above resting parameters in grey matter that are in excellent agreement with literature. Here we demonstrate the method using functionally-defined regions-of-interest in the context of an activation study. We applied the method under high and low signal-to-noise conditions, corresponding respectively to a robust visual stimulus and a modified Stroop task. The estimates fall within the physiological range of literature values, showing the general validity of the GCM approach to yield non-invasively an extensive array of relevant vascular and metabolic parameters.

Task-related BOLD responses and resting-state functional connectivity during physiological clamping of end-tidal CO(2).

Carbon dioxide (CO(2)), a potent vasodilator, is known to have a significant impact on the blood-oxygen level dependent (BOLD) signal. With the growing interest in studying synchronized BOLD fluctuations during the resting state, the extent to which the apparent synchrony is due to variations in the end-tidal pressure of CO(2) (PETCO(2)) is an important consideration. CO(2)-related fluctuations in BOLD signal may also represent a potential confound when studying task-related responses, especially if breathing depth and rate are affected by the task. While previous studies of the above issues have explored retrospective correction of BOLD fluctuations related to arterial PCO(2), here we demonstrate an alternative approach based on physiological clamping of the arterial CO(2) level to a near-constant value. We present data comparing resting-state functional connectivity within the default-mode-network (DMN), as well as task-related BOLD responses, acquired in two conditions in each subject: 1) while subject's PETCO(2) was allowed to vary spontaneously; and 2) while controlling subject's PETCO(2) within a narrow range. Strong task-related responses and areas of maximal signal correlation in the DMN were not significantly altered by suppressing fluctuations in PETCO(2). Controlling PETCO(2) did, however, improve the performance of retrospective physiological noise correction techniques, allowing detection of additional regions of task-related response and resting-state connectivity in highly vascularized regions such as occipital cortex. While these results serve to further rule out systemic physiological fluctuations as a significant source of apparent resting-state network connectivity, they also demonstrate that fluctuations in arterial CO(2) are one of the factors limiting sensitivity in task-based and resting-state fMRI, particularly in regions of high vascular density. This must be considered when comparing subject groups who might exhibit differences in respiratory physiology or breathing patterns.

Levodopa influences striatal activity but does not affect cortical hyper-activity in Parkinson's disease.

Motor studies of Parkinson's disease (PD) have shown cortical hypo-activity in relation to nigrostriatal dopamine depletion. Cognitive studies also identified increased cortical activity in PD. We have previously suggested that the hypo-activity/hyper-activity patterns observed in PD are related to the striatal contribution. Tasks that recruit the striatum in control participants are associated with cortical hypo-activity in patients with PD, whereas tasks that do not result in cortical hyper-activity. The putamen, a structure affected by the neurodegeneration observed in PD, shows increased activation for externally-triggered (ET) and self-initiated (SI) movements. The first goal of this study was to evaluate the effect of levodopa on the putamen's response to ET and SI movements. Our second goal was to assess the effect of levodopa on the hypo-activity/hyper-activity patterns in cortical areas. Patients with PD on and off levodopa and healthy volunteers performed SI, ET and control finger movements during functional magnetic resonance imaging. Healthy participants displayed significant differences in putamen activity in ET and SI movements. These differences were reduced in patients off medication, with non-task-specific increases in activity after levodopa administration. Furthermore, the ventrolateral prefrontal cortex showed significant increases in activity during SI movements in healthy controls, whereas it was hypo-active in PD. This region showed significantly increased activity during ET movements in patients off medication. Levodopa had no effect on this discrepancy. Our results suggest that dopamine replacement therapy has a non-task-specific effect on motor corticostriatal regions, and support the hypothesis that increases and decreases in cortical activity in PD are related to the mesocortical dopamine pathway imbalance.

Elimination of visually evoked BOLD responses during carbogen inhalation: implications for calibrated MRI.

Breathing a mixture of 10% CO(2) with 90% O(2) (referred to here as carbogen-10) increases blood flow due to the vasodilatory effect of CO(2), and raises blood O(2) saturation due to the enriched oxygen level. These effects both tend to reduce the level of deoxygenated hemoglobin in brain tissues, thereby reducing the potential for further increases in BOLD contrast. In the present study, blocks of intense visual stimulation (60s) were presented amid longer blocks (180s) during which subjects breathed various fractional concentrations (0-100%) of carbogen-10 diluted with medical air. When breathing undiluted carbogen-10, the BOLD response to visual stimulation was reduced below the level of noise against the background of the carbogen-10 response. At these concentrations, the total (visual+carbogen) BOLD response amplitude (7.5±1.0%, n=6) converged toward that seen with carbogen alone (7.5±1.0%, n=6). In spite of the almost complete elimination of the visual BOLD response, pseudo-continuous arterial spin-labeling on a separate cohort indicated a largely preserved perfusion response (89±34%, n=5) to the visual stimulus during inhalation of carbogen-10. The previously discussed observations suggest that venous saturation can be driven to very high levels during carbogen inhalation, a finding which has significant implications for calibrated MRI techniques. The latter methods involve estimation of the relative change in venous O(2) saturation by expressing activation-induced BOLD signal increases as a fraction of the maximal BOLD signal M that would be observed as venous saturation approaches 100%. While the value of M has generally been extrapolated from much smaller BOLD responses induced using hypercapnia or hyperoxia, our results suggest that these effects could be combined through carbogen inhalation to obtain estimates of M based on larger BOLD increases. Using a hybrid BOLD calibration model taking into account changes in both blood flow and arterial oxygenation, we estimated that inhalation of carbogen-10 led to an average venous saturation of 91%, allowing us to compute an estimated M value of 9.5%.

Complex organic matter in Titan's atmospheric aerosols from in situ pyrolysis and analysis.

Aerosols in Titan's atmosphere play an important role in determining its thermal structure. They also serve as sinks for organic vapours and can act as condensation nuclei for the formation of clouds, where the condensation efficiency will depend on the chemical composition of the aerosols. So far, however, no direct information has been available on the chemical composition of these particles. Here we report an in situ chemical analysis of Titan's aerosols by pyrolysis at 600 degrees C. Ammonia (NH3) and hydrogen cyanide (HCN) have been identified as the main pyrolysis products. This clearly shows that the aerosol particles include a solid organic refractory core. NH3 and HCN are gaseous chemical fingerprints of the complex organics that constitute this core, and their presence demonstrates that carbon and nitrogen are in the aerosols.

Solvent extraction of organic molecules of exobiological interest for in situ analysis of the Martian soil.

A solid-liquid extraction method able to perform in situ extraction of organic compounds on Mars is proposed. The extraction efficiency of various organic solvents was tested and compared to that of water. The selected key compounds are molecules of exobiological interest: glycine, alanine, serine, glutamic acid, oxalic acid, benzoic acid, phthalic acid, isophthalic acid, terephthalic acid and 1,3,5-benzenetricarboxylic acid. Among the organic solvents, propanol gives the highest yield of extraction for all the targeted compounds except for benzoic acid. A mixture of propanol and ethyl acetate increases significantly the extraction yield of benzoic acid. The extraction time was considerably reduced (140 h to 15 min) by using sonication. The method is discussed for an easy automation with coupling to an in situ GC-MS space instrument.

Gas chromatography for in situ analysis of a cometary nucleus. IV. Study of capillary column robustness for space application.

As part of the development of the European Space Agency Rosetta space mission to investigate a cometary nucleus, the selection of columns dedicated to the gas chromatographic subsystem of the Cometary Sampling and Composition (COSAC) experiment was achieved. Once the space probe launched, these columns will be exposed to the harsh environmental constraints of space missions: vibrations, radiation (by photons or energetic particles), space vacuum, and large temperature range. In order to test the resistance of the flight columns and their stationary phases, the columns were exposed to these rough conditions reproduced in the laboratory. The comparison of the analytical performances of the columns, evaluated prior and after the environmental tests, demonstrated that all the columns withstand space constraints, and that their analytical properties were preserved. Therefore, all the selected capillary columns, even having porous layer or chiral stationary phases, were qualified for space exploration.

Gas chromatography for in situ analysis of a cometary nucleus III. Multi-capillary column system for the cometary sampling and composition experiment of the Rosetta lander probe.

The cometary sampling and composition (COSAC) experiment is one of the principal experiments of the surface lander probe of the European Space Agency Rosetta mission to be launched in January 2003. The instrument is designed for the in situ chemical analysis of a cometary nucleus as the details of the nucleus composition are of primary importance for understanding both the formation of the solar system, and the origin of life on Earth. The COSAC experiment consists of an evaporation/pyrolysis device and two analytical systems: a multi-column gas chromatograph and a high-resolution time-of-flight mass spectrometer which may either be operated alone or in a coupled mode. The gas chromatograph includes five general purpose chromatographic columns and three chiral ones, all mounted in parallel. Taking into account the chemical species potentially present in the cometary nucleus as well as the space constraints, a set of five complementary columns was selected to perform the separation and identification of the compounds present in the cometary nucleus. This set of columns includes a carbon molecular sieve porous-layer open tubular (PLOT) column used for the separation of both the noble and other permanent gases, and the C1-C2 hydrocarbons. A second PLOT column uses a divinylbenzene-ethylene glycol-dimethylacrylate porous polymer as stationary phase for the analysis of a wide range of C1-C2 organic molecules, Two complementary wall-coated open tubular (WCOT) columns with polydimethylsiloxane (PDMS) liquid stationary phases, one containing cyanopropyl-phenylsiloxane and the other diphenylsiloxane groups, are designed to target the same range of organic compounds (C3-C7) which could be representative of the widest range of cometary compounds. A third WCOT column with an apolar stationary phase made of non-substituted PDMS is used for the separation and identification of higher-molecular-mass compounds (up to C10) and aromatic species (monoaromatic and polyaromatic). This paper describes these five general-purpose capillary PLOT and WCOT columns, selected to be used in the COSAC GC system. The analytical capabilities are examined with a special emphasis on the exobiological and planetological implications.

Detection of martian amino acids by chemical derivatization coupled to gas chromatography: in situ and laboratory analysis.

If there is, or ever was, life in our solar system beyond the Earth, Mars is the most likely place to search for. Future space missions will have then to take into account the detection of prebiotic molecules or molecules of biological significance such as amino acids. Techniques of analysis used for returned samples have to be very sensitive and avoid any chemical or biological contamination whereas in situ techniques have to be automated, fast and low energy consuming. Several possible methods could be used for in situ amino acid analyses on Mars, but gas chromatography would likely be the most suitable. Returned samples could be analyzed by any method in routine laboratory use such as gas chromatography, already successfully performed for analyses of organic matter including amino acids from martian meteorites. The derivatization step, which volatilizes amino acids to perform both in situ and laboratory analysis by gas chromatography, is discussed here.

Chemical derivatization of amino acids for in situ analysis of Martian samples by gas chromatography.

Three different methods of derivatization are tested in order to select and optimize one for the in situ analysis of amino acids in Martian samples. The silylation procedure can easily be automated with a high yield and a linear response in a large range of concentrations. The alkylation method is simple and easily automated, but irreproducible data are obtained for the reaction in the GC liner at quite a high temperature (300 degrees C). Moreover by-products of the reaction interfere in the GC chromatograms and mass spectrometry detection is needed for product identification. The chloroformate derivatization has several advantages such as one-step reaction and short time analysis. The main problem of this procedure is the shaking step which difficult to develop in space application.

Study of the retention properties of warfarin enantiomers on a beta-cyclodextrin polymeric support.

High-performance liquid chromatography was used to study the retention properties of (R)- and (S)-warfarins on a silica support coated with a beta-cyclodextrin polymer. The influence of the methanol content of the acetate buffer eluent was investigated at pH 4. The measure of the variations of retention time with temperature enables one to determine the enthalpy and the entropy of adsorption. The plot of the two thermodynamic functions shows a minimum around 30% (v/v) methanol. At low methanol contents, the decrease of the hydrophobic interactions with increasing methanol content explains the decrease of the enthalpic and entropic terms. Above 40% (v/v) methanol, the decrease of the adsorption enthalpy absolute value is due to the solvation by the organic component. From the analysis of peak shape in mass-overload conditions, the column capacity toward each enantiomer was determined. A lower capacity was found toward (S)-warfarin, the more retained enantiomer. Peak shape analysis in mass-overload conditions was used to determine the adsorption isotherm. A Langmuir-type adsorption isotherm accounts well for the experimental data.

Analysis of complex mixtures recovered from space missions statistical approach to the study of Titan atmosphere analogues (tholins).

To study Titan, the largest moon of Saturn, laboratory simulation experiments have been performed to obtain analogues of Titan's aerosols (named tholins) using different energy sources. Tholins, which have been demonstrated to represent aerosols in Titan's haze layers, are a complex mixture, resulting from the chemical evolution of several hydrocarbons and nitriles. Their chromatographic analysis yields complex chromatograms, which require the use of mathematical procedures to extract from them all the information they contain. Two different chemometric approaches (the Fourier analysis approach and the statistical model of peak overlapping) have been successfully applied to pyrolysis-GC-MS chromatogram of a tholin sample. Fundamental information on the mixture's chemical composition (number of components, m) and on the separation system performance (separation efficiency, sigma) can be easily estimated: the excellent correspondence between the data calculated by the two independent procedures proves the reliability of the statistical approaches in characterizing a tholin chromatogram. Moreover, the plot of autocorrelation function contains, in a simplified form, all the information on the retention pattern: retention recursivities can be easily singled out and related to specific molecular structure variations. Therefore, the autocorrelation function (ACF) plot constitutes a simplified fingerprint of the pyrolysis products of tholins, which can be used as a powerful tool to characterize a tholin sample.

Gas chromatography for in situ analysis of a cometary nucleus. II. Analysis of permanent gases and light hydrocarbons with a carbon molecular sieve porous layer open tubular column.

Considering the severe constraints of space instrumentation, a great improvement for the in situ gas chromatographic (GC) determination of permanent and noble gases in a cometary nucleus is the use of a new carbon molecular sieve porous layer open tubular (PLOT) column called Carbobond. No exhaustive data dealing with this column being available, studies were carried out to entirely characterize its analytical performances, especially when used under the operating conditions of the cometary sampling and composition (COSAC) experiment of the European Space Agency (ESA) Rosetta space mission to be launched in 2003 for a rendezvous with comet 46 P/Wirtanen in 2011. The high efficiency and speed of analysis of this column at both atmospheric and vacuum outlet column pressure is demonstrated, and the kinetic mass transfer contribution of this carbon molecular sieve adsorbent is calculated. Besides, differential adsorption enthalpies of several gases and light hydrocarbons were determined from the variation of retention volume with temperature. The data indicate close adsorption behaviors on the Carbobond porous layer adsorbent and on the carbon molecular sieve Carboxen support used to prepare the packed columns. Moreover, taking into account the in situ operating conditions of the experiment, a study of two columns with different porous layer thicknesses allowed one to optimize the separation of the target components and to select the column parameters compatible with the instrument constraints. Comparison with columns of similar selectivity shows that these capillary columns are the first ones able to perform the same work as the packed and micro-packed columns dedicated to the separation of this range of compounds in GC space exploration.

Characterization of Human Serum Albumin Adsorbed on a Porous Anion-Exchange Support.

Fourier transform infrared (FTIR) spectroscopy and high-performance liquid chromatography (HPLC) were used to study the adsorption behavior of human serum albumin (HSA) onto a polymer-layer type anion exchanger, poly(vinylimidazole) (PVI) adsorbed and cross-linked on a porous silica support. The data are compared with previous results for HSA adsorbed on C6 alkyl chains grafted on the same silica matrix. For the adsorption onto the PVI support from a phosphate buffer solution of low ionic strength (pD 7.4), the FTIR experiments reveal only very weak structural and solvation changes. A large fraction of the protein remains irreversibly adsorbed and the amount retained at equilibrium is close to that observed for the adsorption on the reversed-phase support, although the structural effect of the stationary phase was much larger with the grafted C6 alkyl chains. Comparing to the solution state, only 2% of the HSA backbone is modified by adsorption on PVI, whereas 12% alterations are involved for the protein adsorbed on the reversed-phase support. When adsorbed from an eluent containing 20% acetonitrile, the amount of HSA retained by the PVI ion exchanger is about twice that measured with the buffer alone. This result is explained by a more compact structure of the protein when dissolved in the organoaqueous solvent. The presence of acetonitrile does not markedly affect the elution front of HSA adsorbed on the charged hydrophilic adsorbent. This result contrasts with the much lower apparent adsorption rate observed when HSA is adsorbed on the grafted alkyl chain support in the presence of acetonitrile in the buffer. Copyright 1998 Academic Press.

Capillary electrophoretic behavior of milk proteins in the presence of non-ionic surfactants.

The electrophoretic behavior of alpha-lactalbumin and beta-lactoglobulins (A and B) in the presence of non-ionic surfactants was studied by capillary electrophoresis (CE), using a poly(ethylene glycol) coated capillary column. The surfactants (Tween 20, Brij 35 and 78) were used as buffer additives. The separation is based on the difference in the strength of protein-surfactant association complexes, which results in a change of the effective electrophoretic mobility. The modification of the electrophoretic mobilities of proteins was observed and this variation permitted the estimation of the interaction between protein and surfactant. The effect of surfactant type and concentration on the migration behavior of protein in CE is discussed. It is found that the retention behavior of the milk proteins (the alpha-lactalbumin and the beta-lactoglobulins) in CE is very different. The pH of the buffer and the surfactant type influence significantly the protein-surfactant interactions.

Study of conformational effects of recombinant interferon gamma adsorbed on a non-porous reversed-phase silica support.

Reversed-phase chromatography is a powerful method for separating recombinant interferon gamma and one of its analogues differing only by a single amino acid residue. Structural differences of the proteins explain this separation ability as demonstrated from adsorption studies on a non-porous reversed-phase support. To reveal the structural differences occurring in the adsorbed state, two different and independent methods were employed. The variation of the retention with the slope of the linear gradient gave information about the molecular contact area of the protein with the support. For different experimental conditions, these data were correlated with the adsorbent capacities measured on an n-octadecyl-modified non-porous silica support. These supports are useful for these types of experiments because the protein is adsorbed exclusively at the external surface of the beads. Moreover, a small amount of protein is necessary to saturate the column, owing to its low capacity.

Molecular approach to protein-polymer interactions in ion-exchange chromatography.

A model was developed and implemented to aid in understanding and predicting the retention behaviour of proteins in ion-exchange chromatography. The model structures chosen were calcium-loaded and -depleted alpha-lactalbumin (ALC) and hen egg white lysozyme (HEWL) and a comparison was made with chromatographic measurements. A characteristic charge of -3.4 was found under the experimental conditions applied for both forms of ALC, and HEWL was not retained. The model explicitly considers all of the atoms, each being assigned a set of force field parameters. Because of the computational time necessary to include them, water molecules were not taken into account, but a sigmoidal function of the dielectric permittivity was introduced in the calculations. Interaction potential energies from bulk down to the contact were evaluated for each protein. The results were in qualitative agreement with those of the chromatographic experiments. It was possible to reproduce the difference in retention between both forms of ALC and also the behaviour of HEWL.

Chromatographic kinetic measurements of human serum albumin adsorption on monoclonal antibodies.

A chromatographic method was employed to study the kinetics of human serum albumin (HSA) adsorbed on immobilized monoclonal antibodies. The antibodies of various specificities were covalently bound to a high-performance liquid chromatography (HPLC) silica support. For very low desorption rates, successive amounts of the reacting protein were injected until column saturation. The analysis of the increase of the non-retained fraction calculated from peak area measurements gives the capacity of the support and the rate of the biospecific adsorption process. The model is based on a second-order Langmuir kinetic law and assumes a global mass transfer for the adsorption process. The use of a silica support of small pore size permits the reduction of the contribution for mass transfer in the stagnant fluid and the decrease in the column capacity: due to its large size, the reacting molecule is adsorbed on the external surface of the particle. The adsorption rate constants of HSA on five monoclonal anti-HSA antibodies of different specificities were determined. For all the immuno-adsorbents studied, the adsorption rate constant is significantly lower than that found on immobilized polyclonal antibodies. Measurements at different flow rates reveal that the mass transfer due to the transport to the adsorbent surface is small and can be estimated.

Kinetic study of the adsorption of human serum albumin on immobilized antibody using the split-peak effect in immunochromatography.

The split-peak effect was used to determine the association rate constant of the antigen-immobilized antibody reaction. The amount of immobilized human serum albumin antibody on the chromatographic support was varied in order to find the optimal conditions to reduce the mass transfer contribution in the stagnant mobile phase fluid and measure the effective association rate constant of human serum albumin with the immobilized antibody. Kinetic studies as a function of flow-rate demonstrate the validity of the method consisting in determining the association rate constant from measurements performed on columns of various capacities. These experiments show that limitations due to mass transfer to the surface of the adsorbent are minimized at high flow-rates and for a low density of immobilized ligand.