Total Capillary Volume Research Articles

Sidestream dark field imaging and biomarker evaluation reveal minimal significant changes to the microcirculation and glycocalyx in a canine hemorrhagic shock model.

To investigate the effects of hemorrhagic shock and fresh whole blood resuscitation on the microcirculation and endothelial glycocalyx using sidestream dark field (SDF) imaging and plasma biomarkers. 8 purpose-bred dogs. Pressure-targeted hemorrhagic shock was induced in anesthetized dogs. SDF measurement of perfused boundary region and microcirculatory variables (RBC flow, total vessel density, and relative and absolute capillary blood volume), biomarker measurement (heparan sulfate, hyaluronan, VE-cadherin, and syndecan-1), mean arterial blood pressure, and cardiac output measurement were performed before anesthesia (TP0), after induction (TP1), after hemorrhagic shock (TP2), and after 50% retransfusion (TP3) and 100% retransfusion (TP4). At TP1, TP2, TP3, and TP4, mean arterial blood pressure was 74.25 ± 7.17 mm Hg, 49.50 ± 13.74 mm Hg, 63.50 ± 13.29 mm Hg, and 71.38 ± 8.77 mm Hg, and cardiac output was 2.57 ± 1.01 L/min, 0.8 ± 0.36 L/min, 1.81 ± 0.57 L/min, and 2.93 ± 1.22 L/min, respectively. Heparan sulfate, hyaluronan, syndecan-1, and VE-cadherin ranges were 24.80 to 77.72 ng/mL, 5.77 to 105.06 ng/mL, below detection to 1,545.69 pg/mL, and 0 to 2.52 ng/mL, respectively. Perfused boundary region, RBC flow, total vessel density, and relative and absolute capillary blood volume ranges were 1.75 to 2.68 µm, 89.6 to 584.5 µm/s, 51.7 to 1,914.3 mm/m2, 0.94 to 1.53 103 μm3, and 1.50 to 94.30 103 μm3, respectively. Heparan sulfate decreased significantly over time (P = .016). No significant differences were found for microcirculatory variables, perfused boundary regions, or other biomarkers. This was the first study to assess microvascular dysfunction and endothelial shedding in a canine hemorrhagic shock model using SDF microscopy (Glycocheck) and plasma biomarkers. Further studies are needed to determine clinical relevance.

Capillary Electrophoresis-Mass Spectrometry for the Direct Analysis of Metabolites in Highly Saline Samples Using In-Capillary Preconcentration.

Capillary electrophoresis-mass spectrometry (CE-MS) is gaining interest for metabolomics studies because of its high separation efficiency, selectivity, and versatility. The ability to inject nanoliters from only a few microliters of sample in the injection vial makes this approach very suited for volume-limited applications. However, the low injection volumes could compromise the detection sensitivity of CE-MS, thereby potentially limiting its scope in metabolomics. To overcome this issue, online sample preconcentration methods have been developed to increase sample-loading volumes without hampering the intrinsic high separation efficiency of CE. In this protocol, online preconcentration with sample stacking based on pH junction was assessed for the direct profiling of endogenous metabolites in rat brain microdialysates. Sample stacking was realized by a pre-injection of ammonium hydroxide, followed by a large sample injection (i.e., about 17% of the total capillary volume). It is shown that this relatively simple and fast preconcentration procedure is fully compatible with the high-salt concentration in microdialysates and significantly improves the detection sensitivity of the CE-MS method.

CEToolbox: Specialized calculator for capillary electrophoresis users as an android application.

CE has been demonstrated to be a useful and powerful separation method for the characterization of charged and neutral molecules. Since the end of the 1980s and the development of the first commercialized CE device, the use of this separation method has continued to grow for academic and industrial research involving inexorably increasing of the number of CE users. Whatever the application domain, each CE user is daily confronted to the same problems often based on basic calculations of separation properties. In order to help the community of CE users to get quickly and easily a lot of information, and desiring to provide a tool running on mobile platforms, CEToolbox has been developed as a free Android application. Within few clicks, CEToolbox offers extensive injection information as injected volume, total capillary volume, proportion and amount of injected sample, rinsing time, and electrical field. Moreover, three additional tabs allow to obtain the calculation of the viscosity and the conductivity of BGE, and the separation flow rates. Finally, a last tab is dedicated to the calculation of electroosmotic mobility and effective mobilities for a maximum of 20 compounds. CEToolbox, which can be downloaded for free on Google and F-Droid application stores, was developed to simplify the daily of CE users regardless of the CE devices.

Sprouting Angiogenesis: A Numerical Approach with Experimental Validation.

A functional vascular network is essential to the correct wound healing. In sprouting angiogenesis, vascular endothelial growth factor (VEGF) regulates the formation of new capillaries from pre-existing vessels. This is a very complex process and mathematical formulation permits to study angiogenesis using less time-consuming, reproducible and cheaper methodologies. This study aimed to mimic the chemoattractant effect of VEGF in stimulating sprouting angiogenesis. We developed a numerical model in which endothelial cells migrate according to a diffusion-reaction equation for VEGF. A chick chorioallantoic membrane (CAM) bioassay was used to obtain some important parameters to implement in the model and also to validate the numerical results. We verified that endothelial cells migrate following the highest VEGF concentration. We compared the parameters-total branching number, total vessel length and branching angle-that were obtained in the in silico and the in vivo methodologies and similar results were achieved (p-value smaller than 0.5; n = 6). For the difference between the total capillary volume fractions assessed using both methodologies values smaller than 15% were obtained. In this study we simulated, for the first time, the capillary network obtained during the CAM assay with a realistic morphology and structure.

Direct profiling of endogenous metabolites in rat brain microdialysis samples by capillary electrophoresis-mass spectrometry with on-line preconcentration

Metabolic profiling of body fluids from small animal models is often used in (translational) biological studies in order to obtain insight into the underlying molecular mechanisms of (complex) diseases. An example is the use of brain microdialysis samples from rats to study neurological disorders by means of a metabolomics approach. From an analytical point of view, the profiling of (endogenous) metabolites in rat brain microdialysates is challenging because of the limited sample volume for both sample preparation and injection, notably in longitudinal studies. In this work, we have assessed the analytical performance of capillary electrophoresis-mass spectrometry (CE-MS) for the direct profiling of endogenous metabolites in rat brain microdialysates, i.e. without using any sample preparation or derivatization. An on-line preconcentration procedure with sample stacking, which was fully compatible with the high-salt concentration in microdialysates, was used to significantly improve the detection sensitivity of the CE-MS method for metabolic profiling. A response surface methodology, applying a Box-Behnken design, was considered to determine the optimal conditions for preconcentration. A linear response (R2>0.99) for selected metabolites in the concentration range from 0.05 to 10 µM was obtained in perfusate samples. Interday RSD values for peak area and migration time were 2.6-19% and below 3.8%, respectively. Limits of detection ranged from 11 to 284 nM when employing an injection volume of about 291 nL, corresponding to 17% of the total capillary volume. The utility of the CE-MS approach was demonstrated by the direct profiling of endogenous metabolites in rat brain microdialysates. At least 48 compounds could be analyzed of which 25 were provisionally identified and quantified.

Short-term post-implantation dynamics of in vitro engineered human microvascularized adipose tissues

Engineered adipose tissues are developed for their use as substitutes for tissue replacement in reconstructive surgery. To ensure a timely perfusion of the grafted substitutes, different strategies can be used such as the incorporation of an endothelial component. In this study, we engineered human adipose tissue substitutes comprising of functional adipocytes as well as a natural extracellular matrix using the self-assembly approach, without the use of exogenous scaffolding elements. Human microvascular endothelial cells (hMVECs) were incorporated during tissue production in vitro and we hypothesized that their presence would favor the early connection with the host vascular network translating into functional enhancement after implantation into nude mice in comparison to the substitutes that were not enriched in hMVECs. In vitro, no significant differences were observed between the substitutes in terms of histological aspects. After implantation, both groups presented numerous adipocytes and an abundant matrix in addition to the presence of host capillaries within the grafts. The substitutes thickness and volume were not significantly different between groups over the short-term time course of 14 days (d). For the microvascularized adipose tissues, human CD31 staining revealed a human capillary network connecting with the host microvasculature as early as 3 d after grafting. The detection of murine red blood cells within human CD31+ structures confirmed the functionality of the human capillary network. By analyzing the extent of the global vascularization achieved, a tendency towards increased total capillary network surface and volume was revealed for prevascularized tissues over 14 d. Therefore, applying this strategy on thicker reconstructed adipose tissues with rate-limiting oxygen diffusion might procure added benefits and prove useful to provide voluminous substitutes for patients suffering from adipose tissue loss or defects.

Online Preconcentration in Capillaries by Multiple Large-Volume Sample Stacking: An Alternative to Immunoassays for Quantification of Amyloid Beta Peptides Biomarkers in Cerebrospinal Fluid.

A novel electrokinetic preconcentration approach, so-called multiple pressure-assisted large-volume sample stacking with an electroosmotic flow pump (M-PA-LVSEP), was developed to allow in-capillary enrichment and separation of analytes from unlimited sample volumes. With this approach, the inherent limitation of in-capillary electrokinetic preconcentrations to the separation capillary volume can be overcome. The M-PA-LVSEP protocol relies on repeated cycles of pressure-assisted electroosmotic pumping and injection of extremely large sample volumes for analyte stacking and sample matrix removal. This technique was developed to address the challenge of sensitive and simultaneous determination of several amyloid β (Aβ) peptides, which are biomarkers for the molecular diagnosis of Alzheimer's disease (AD). For the first time, reliable quantification of different species of fluorescently derivatized Aβ peptides, that is, Aβ 1-42, Aβ 1-40, and Aβ 1-38 down to subnanomolar ranges in cerebrospinal fluids (CSF) from AD and non-demented patients (healthy controls) was made possible without recourse to immunoassay, immunoprecipitation, or mass spectrometry approaches. Based on the stacking from a sample plug representing up to 400% of the total capillary volume, sensitive enhancement factors up to 170 could be achieved with this "antibody free" approach. Quantification limits for these Aβ peptides down to 0.05 nM with capillary electrophoresis coupled with laser-induced fluorescent detection could be obtained. Excellent agreement between results from M-PA-LVSEP and the gold standard ELISA method was achieved for measurements of Aβ 1-42 in CSF, with a determination correlation (r2) better than 0.993.

Adsorption and movement of water by skin of the Australian thorny devil (Agamidae: Moloch horridus).

Moisture-harvesting lizards, such as the Australian thorny devil Moloch horridus, have remarkable adaptations for inhabiting arid regions. Their microstructured skin surface, with channels in between overlapping scales, enables them to collect water by capillarity and passively transport it to the mouth for ingestion. We characterized this capillary water transport for live thorny devils using high-speed video analyses. Comparison with preserved specimens showed that live lizards are required for detailed studies of skin water transport. For thorny devils, there was no directionality in cutaneous water transport (unlike Phrynosoma) as 7 µl water droplets applied to the skin were transported radially over more than 9.2 mm. We calculated the total capillary volume as 5.76 µl cm−2 (dorsal) and 4.45 µl cm−2 (ventral), which is reduced to 50% filling by the time transportation ceases. Using micro-computed tomography and scanning electron microscopy of shed skin to investigate capillary morphology, we found that the channels are hierarchically structured as a large channel between the scales that is sub-divided by protrusions into smaller sub-capillaries. The large channel quickly absorbs water whereas the sub-capillary structure extends the transport distance by about 39% and potentially reduces the water volume required for drinking. An adapted dynamics function, which closely reflects the channel morphology, includes that ecological role.

Integration of the free liquid membrane into electrokinetic supercharging – capillary electrophoresis for the determination of cationic herbicides in environmental water samples

A new approach based on the integration of the free liquid membrane (FLM) into electrokinetic supercharging (EKS) was demonstrated to be a new powerful tool used in order to enhance online preconcentration efficiency in capillary electrophoresis (CE). A small plug of water immiscible organic solvent was used as a membrane interface during the electrokinetic sample injection step in EKS in order to significantly enhance the analyte stacking efficiency. The new online preconcentration strategy was evaluated for the determination of paraquat and diquat present in the environmental water samples. The optimised FLM-EKS conditions employed were as follows: hydrodynamic injection (HI) of 20mM potassium chloride as leading electrolyte at 50mbar for 75s (3% of the total capillary volume) followed by the HI of tris(2-ethylhexyl) phosphate (TEHP) as FLM at a 1mm length (0.1% of the capillary volume). The sample was injected at 10kV for 360s, followed by the HI of 20mM cetyl trimethylammonium bromide (CTAB) as terminating electrolyte at 50mbar for 50s (2% of the total capillary volume). The separation was performed in 12mM ammonium acetate and 30mM NaCl containing 20% MeOH at +25kV with UV detection at 205nm. Under optimised conditions, the sensitivity was enhanced between 1500- and 1866-fold when compared with the typical HI at 50mbar for 50s. The detection limit of the method for paraquat and diquat was 0.15–0.20ng/mL, with RSDs below 5.5%. Relative recoveries in spiked river water were in the range of 95.4–97.5%. A comparison was also made between the proposed approach with sole preconcentration of the field-enhanced sample injection (FASI) and EKS in the absence of the FLM.

Variation in the origin of lateral circumflex femoral artery – A case report

Previous work shows that mammary uptake of milk precursors from blood can be affected by the rate of blood flow (F) to the glands. The purpose of the current work was to test the ability of compartmental and cylindrical capillary models to account for the variation in mammary extraction and net uptake of plasma metabolites produced by perturbation of mammary F. The data for model fitting were obtained from a previous experiment in which mammary arteriovenous differences of acetate + β-hydroxybutyrate (2C), glucose, triacylglycerol (TAG), and long-chain fatty acids (LCFA) were measured in 4 cows before, during, and after intraarterial infusion of inhibitors of endothelial nitric oxide synthase and cyclooxygenase, which are 2 major systems of F control in the mammary glands. The 4 models tested were (1) constant extraction within each cow, (2) clearance from an extracellular compartment is a linear function of F with an intercept, (3) total capillary volume in a cylindrical representation is a linear function of F with an intercept, and (4) uptake from an extracellular compartment obeys Henri-Michaelis-Menten kinetics, where maximum velocity (Vmax) is a linear function of F with an intercept. According to prediction errors, model 4 fitted 2C extraction data best, accounting for 82% of the observed variation. The estimated Km (Henri-Michaelis-Menten constant) for venous 2C was 0.4 mM. For glucose clearance, a variant of model 2 with a positive effect of 2C uptake on clearance was identified as best, producing a coefficient of determination (R2) of 0.31. For TAG, model 2 with a positive effect of arterial TAG concentration on TAG clearance was best, with an R2 of 0.22. For LCFA, model 2 with a positive effect of arterial LCFA on LCFA clearance was best, with an R2 of 0.29. Models 2 and 3 fitted the extraction data with the same R2-values and prediction errors, so both compartmental and cylindrical approaches to describing the vascular bed were equally capable of describing the effect of F on mammary uptakes. A combined fit of all best-fit models to extraction data for all 4 metabolites at once explained 52, 42, 73, and 77% of variation in net uptakes of 2C, glucose, TAG, and LCFA, respectively. According to the fitted model, each 1 L/min increase in F increased the mammary volumes of distribution of 2C, glucose, TAG, and LCFA by 13, 14, 18, and 7%, respectively.

Electrokinetic supercharging in nonaqueous capillary electrophoresis for online preconcentration and determination of tamoxifen and its metabolites in human plasma

An online preconcentration method, namely electrokinetic supercharging (EKS), was evaluated for the determination of tamoxifen and its metabolites in human plasma in nonaqueous capillary electrophoresis with ultraviolet detection (NACE-UV). This method was comprehensively optimized in terms of the leading electrolyte (LE) and terminating electrolyte (TE) injection lengths, as well as electrokinetic sample injection time. The optimized EKS conditions employed were as follows: hydrodynamic injection (HI) of 10mM potassium chloride as LE at 150mbar for 36s (4% of total capillary volume). The sample was injected at 10kV for 300s, followed by HI of 10mM pimozide as TE at 150mbar for 36s (4% of total capillary volume). Separation was performed in 7.5mM deoxycholic acid sodium salt, 15mM acetic acid and 1mM 18-crown-6 in 100% methanol at +25kV with UV detection at 205nm. Under optimized conditions, the sensitivity was enhanced between 160- and 600-fold when compared with our previously developed method based on HI at 150mbar for 12s. The detection limit of the method for tamoxifen and its metabolites were 0.05–0.25ng/mL, with RSDs between 2.1% and 3.5%. Recoveries in spiked human plasma were 95.6%–99.7%. A comparison was also made between the proposed EKS approach and the standard field-amplified sample injection (FASI) technique. EKS proved to be 3–5 times more sensitive than the FASI. The new EKS method was applied to the analysis of tamoxifen and its metabolites in plasma samples from breast cancer patients after liquid–liquid extraction.

Predicting extraction and uptake of arterial energy metabolites by the mammary glands of lactating cows when blood flow is perturbed

Previous work shows that mammary uptake of milk precursors from blood can be affected by the rate of blood flow (F) to the glands. The purpose of the current work was to test the ability of compartmental and cylindrical capillary models to account for the variation in mammary extraction and net uptake of plasma metabolites produced by perturbation of mammary F. The data for model fitting were obtained from a previous experiment in which mammary arteriovenous differences of acetate + β-hydroxybutyrate (2C), glucose, triacylglycerol (TAG), and long-chain fatty acids (LCFA) were measured in 4 cows before, during, and after intraarterial infusion of inhibitors of endothelial nitric oxide synthase and cyclooxygenase, which are 2 major systems of F control in the mammary glands. The 4 models tested were (1) constant extraction within each cow, (2) clearance from an extracellular compartment is a linear function of F with an intercept, (3) total capillary volume in a cylindrical representation is a linear function of F with an intercept, and (4) uptake from an extracellular compartment obeys Henri-Michaelis-Menten kinetics, where maximum velocity (Vmax) is a linear function of F with an intercept. According to prediction errors, model 4 fitted 2C extraction data best, accounting for 82% of the observed variation. The estimated Km (Henri-Michaelis-Menten constant) for venous 2C was 0.4 mM. For glucose clearance, a variant of model 2 with a positive effect of 2C uptake on clearance was identified as best, producing a coefficient of determination (R2) of 0.31. For TAG, model 2 with a positive effect of arterial TAG concentration on TAG clearance was best, with an R2 of 0.22. For LCFA, model 2 with a positive effect of arterial LCFA on LCFA clearance was best, with an R2 of 0.29. Models 2 and 3 fitted the extraction data with the same R2-values and prediction errors, so both compartmental and cylindrical approaches to describing the vascular bed were equally capable of describing the effect of F on mammary uptakes. A combined fit of all best-fit models to extraction data for all 4 metabolites at once explained 52, 42, 73, and 77% of variation in net uptakes of 2C, glucose, TAG, and LCFA, respectively. According to the fitted model, each 1 L/min increase in F increased the mammary volumes of distribution of 2C, glucose, TAG, and LCFA by 13, 14, 18, and 7%, respectively.

Full antibody primary structure and microvariant characterization in a single injection using transient isotachophoresis and sheathless capillary electrophoresis-tandem mass spectrometry.

Here we report the complete characterization of the primary structure of a multimeric glycoprotein in a single analysis by capillary electrophoresis (CE) coupled to mass spectrometry (MS). CE was coupled to electrospray ionization tandem MS by means of a sheathless interface. Transient isotachophoresis (t-ITP) was introduced in this work as an electrokinetically based preconcentration technique, allowing injection of up to 25% of the total capillary volume. Characterization was based on an adapted bottom-up proteomic strategy. Using trypsin as the sole proteolytic enzyme and data from a single injection per considered protein, 100% of the amino acid sequences of four different monoclonal antibodies could be achieved. Furthermore, illustrating the effectiveness and overall capabilities of the technique, the results were possible through identification of peptides without tryptic miscleavages or posttranslational modifications, demonstrating the potency of the technique. In addition to full sequence coverages, posttranslational modifications (PTMs) were simultaneously identified, further demonstrating the capacity of this strategy to structurally characterize glycosylations as well as faint modifications such as asparagine deamidation or aspartic acid isomerization. Together with the exquisite detection sensitivity observed, the contributions of both the CE separation mechanism and selectivity were essential to the result of the characterization with regard to that achieved with conventional MS strategies. The quality of the results indicates that recent improvements in interfacing CE-MS coupling, leading to a considerably improved sensitivity, allows characterization of the primary structure of proteins in a robust and faster manner. Taken together, these results open new research avenues for characterization of proteins through MS.

The high aerobic capacity of a small, marsupial rat-kangaroo (Bettongia penicillata) is matched by the mitochondrial and capillary morphology of its skeletal muscles

We examined the structure-function relationships that underlie the aerobic capacities of marsupial mammals that hop. Marsupials have relatively low basal metabolic rates (BMR) and historically were seen as 'low energy' mammals. However, the red kangaroo, Macropus rufus (family Macropodidae), has aerobic capacities equivalent to athletic placentals. It has an extreme aerobic scope (fAS) and its large locomotor muscles feature high mitochondrial and capillary volumes. M. rufus belongs to a modern group of kangaroos and its high fAS is not general for marsupials. However, other hopping marsupials may have elevated aerobic capacities. Bettongia penicillata, a rat-kangaroo (family Potoroidae), is a small (1 kg), active hopper whose fAS is somewhat elevated. We examined the oxygen delivery system in its muscles to ascertain links with hopping. An elevated fAS of 23 provided a relatively high maximal aerobic oxygen consumption ( ) in B. penicillata; associated with this is a skeletal muscle mass of 44% of body mass. Ten muscles were sampled to estimate the total mitochondrial and capillary volume of the locomotor muscles. Values in B. penicillata were similar to those in M. rufus and in athletic placentals. This small hopper had high muscle mitochondrial volume densities (7.1-11.9%) and both a large total capillary volume (6 ml kg(-1) body mass) and total capillary erythrocyte volume (3.2 ml kg(-1)). Apparently, a considerable aerobic capacity is required to achieve the benefits of the extended stride in fast hopping. Of note, the ratio of to total muscle mitochondrial volume in B. penicillata was 4.9 ml O(2) min(-1) ml(-1). Similar values occur in M. rufus and also placental mammals generally, not only athletic species. If such relationships occur in other marsupials, a fundamental structure-function relationship for oxygen delivery to muscles likely originated with or before the earliest mammals.

CZE with On-line Micellar Sample Stacking for Determination of Protein Concentration of Biopharmaceuticals

A capillary zone electrophoresis total protein assay was developed and validated in polyethylene oxide (PEO) dynamically coated capillaries. On-line large-volume sample stacking was employed. Protein samples were denatured using SDS and then injected into PEO-filled capillaries. Such treatment enabled injection of a sample volume of ≈8% of the total capillary volume and stacking of protein-SDS molecules at the interface between the sample plug and the PEO plug. Results showed that SDS enhanced the sensitivity not only by protein denaturation but also by forming micelles, in which protein-SDS partitioned. Sensitivity of the method was further enhanced through using capillaries with (tenfold) extended detection pathlength. Such strategies resulted in a limit of detection of 0.26 μg mL−1 (3.64 nM BSA). A linear relationship between protein concentration and integrated peak area was obtained over a wide concentration range (8.49–135.87 μg mL−1—R2 = 0.995). The method is particularly useful for determination of total protein concentration in chromatography fractions. It overcomes low UV absorptivity of proteins, presence of UV absorbing additives and high salt content. Contrary to conventional methods for determination of protein concentration, this method does not involve an interaction with a dye. Thus, variations due to differences in surface properties among proteins or due to differences in posttranslational modifications of the same protein are eliminated. The protocol was successfully applied for the determination of the concentration of a biopharmaceutical protein rhMBP in chromatography fractions. This protein has been previously produced in milk of transgenic cows and several charge isoforms were detected.

A model of perfusion of the healthy human lung

This study presents a model that simulates the pulmonary capillary perfusion. The model describes the lungs as divided into horizontal layers and includes: capillary geometry; capillary wall elasticity; pressure at the pulmonary artery; blood viscosity; the effect of the chest wall; the change in lung height and hydrostatic effects of the lung tissue and of the blood during breathing. The model simulates pulsatile blood perfusion with an increasing blood distribution down the lungs, in agreement with previous experimental studies. Moreover the model is in agreement with experimentally measured total capillary perfusion, total capillary volume, total capillary surface area and transition time of red blood cells passing through the pulmonary capillary network. The presented model is the first to be validated against the mentioned experimental data and to model the link between airway pressure, lung volume and perfusion.

Heart‐cutting 2D‐CE with on‐line preconcentration for the chiral analysis of native amino acids

The use of transient moving chemical reaction boundary (tMCRB) was investigated for the on-line preconcentration of native amino acids in heart-cutting 2D-CE with multiple detection points using contactless conductivity detection. The tMCRB focusing was obtained by using ammonium formate (pH 8.56) as sample matrix and acetic acid (pH 2.3) as a BGE in the first dimension of the heart-cutting 2D-CE. Different experimental parameters such as the injected volume and the concentration in ammonium formate were optimized for improving the sensitivity of detection. A stacked fraction from the first dimension was selected, isolated in the capillary, and then separated in the second dimension in the presence of a chiral selector ((+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid). This on-line tMCRB preconcentration coupled with heart-cutting 2D-CE was applied with success to the chiral separation of D,L-phenylalanine, and D,L-threonine in a mixture of 22 native amino acids. The sample mixture was diluted in 0.8 M of ammonium formate, and injected at a concentration of 2.5 muM for each enantiomer with a volume corresponding to 10% of the total capillary volume. An LOD (S/N=3) of 2 muM was determined for L-threonine.

A Mathematical Physiological Model of the Pulmonary Capillary Perfusion

This study presents a stratified model that simulates the pulmonary capillary blood flow under influence of different lung volumes. The model includes capillary geometry, capillary wall elasticity, pressure exerted by the heart, blood viscosity, the effect of the chest wall and hydrostatic effects of the lung tissue and of the blood. The model simulates highly pulsatile blood flow with a heterogenous flow distribution down the lungs, in agreement with previous experimental studies. Moreover the model is in agreement with experimentally measured total capillary flow, total capillary volume, total capillary surface area and transition time of red blood cells passing through the pulmonary capillary network. The presented model is the first to describe the link between lung volume and perfusion.

Abstract 3726: Right Ventricular Microcirculatory Changes in Pulmonary Hypertension-Associated Right Heart Failure Imaged by Three-Dimensional Confocal Microscopy

Background: Right ventricular (RV) ischemia occurs in pulmonary hypertension (PH) patients despite normal coronary angiograms. O 2 supply-demand balance in the pressure-overloaded RV is determined by the interplay between reduced coronary perfusion (due to systemic hypotension and compression of coronary vessels) and a wall stress-related increase in myocardial O 2 consumption. The role of capillary rarefaction, which is involved in pressure overload induced left heart failure, has not been investigated in PH-associated RV failure. Methods: Rats received a single dose of the VEGF receptor blocker SU5416 and were exposed to 4 wks of normobaric hypoxia and 2 wks of sea level room air. This regimen induces angioproliferative changes in the pulmonary circulation, severe PH and RV failure. At 6 wks, the rats received intravital injections of Texas-Red conjugated tomato lectin, which is an N-acetylglucosamine specific marker of blood vessels in rodents. After circulation of the lectin for 5 min, papaverin was injected to promote maximal vasodilation of all perfused blood vessels. Animals were exsanguinated and perfusion-fixed with paraformaldehyde. After overnight fixation, 300 μm thick transverse sections of the myocardium were bleached in a methanol/DMSO mixture and nuclei were stained using DAPI. Prior to 3-dimensional imaging under a confocal microscope, further tissue clearing was achieved using organic solvents. Results: Z-stacks of 2-dimensional images were made in whole-mount cardiac specimens, using a step size of 0.32 μm. 3-dimensional reconstruction allowed quantification of the total capillary volume, which was reduced in the PH model compared to controls (2.9±1% vs. 5.7±1% of tissue volume, p<0.05). The reconstructions showed a remarkable increased morphological heterogeneity of the capillaries in the failing RV. In contrast, images of the LV microcirculation in the SU5416 treated animals were not different from those of controls. Conclusions: RV failure in experimental PH is associated with capillary rarefaction. 3-dimensional imaging of the myocardial microcirculation can be achieved using intravital injections of tomato lectin and subsequent confocal imaging.

Stacking and separation of urinary porphyrins in capillary electrophoresis: Optimization of concentration efficiency and resolution

We demonstrated that anionic porphyrins could be stacked and separated in micellar electrokinetic chromatography (MEKC) and microemulsion electrokinetic chromatography (MEEKC) by applying acetonitrile and high salt content in human urine sample matrix. The introduction of sample containing acetonitrile and sodium chloride into the CE capillary at more than 10% of the total capillary volume resulted in the improvement of peak resolution and the enhancement of detection sensitivity. The achieved acetonitrile stacking enrichment factors of six porphyrins ranged from 12 to 32 in MEKC and from 28 to 33 in MEEKC, respectively. The stacking technique was successfully applied for analyzing porphyrins present in urine samples that were deproteinized with acetonitrile. For the analysis of coproporphyrin isomers, addition of the sodium cholate (SC) into micelle and microemulsion solutions provided adequate resolution. Calibration curves obtained for the determination of coproporphyrin isomers were found linear between 30 and 400 nmol L −1, and the limit of detection (LOD) was 20 nmol L −1 in MEEKC. Intra- and interday precisions ( n = 11) in the microemulsion separation system for the isomers at spiked concentrations of 40–400 nmol L −1 in urine were in the range of 0.1–0.4% and 0.7–7.6% for migration time and peak area, respectively. Coproporphyrin III, coproporphyrin I and uroporphyrin were detected at levels of 80.7 nmol L −1, 32.3 nmol L −1 and 19.8 nmol L −1, respectively, in the urine samples collected from healthy individuals. Different porphyrin profiles, however, were observed in urine samples from porphyria cutanea tarda (PCT) patients.