High-dose intravenous immunoglobulins: A promising therapeutic approach for idiopathic systemic capillary leak syndrome

The systemic capillary leak syndrome (SCLS), also known as Clarkson’s disease, is a life-threatening disorder of unknown cause. It is characterised by recurrent acute episodes of hypotension, weight gain and generalised oedema with haemoconcentration and hypoproteinaemia caused by paroxysmal capillary hyperpermeability with a shift of plasma fluid from the intravascular to the interstitial space. We report the case of a 40-year-old woman with chronic SCLS treated with high-dose intravenous immunoglobulins, after a prophylactic therapy with theophylline and terbutaline was poorly tolerated and failed to decrease the frequency and severity of the attacks sufficiently.

Enantioselective capillary electrophoresis for the assessment of CYP3A4-mediated ketamine demethylation and inhibition in vitro

Enantioselective CE with sulfated cyclodextrins as chiral selectors was used to determine the CYP3A4-catalyzed N-demethylation kinetics of ketamine to norketamine and its inhibition in the presence of ketoconazole in vitro. Ketamine, a chiral phencyclidine derivative, was incubated with recombinant human CYP3A4 from a baculovirus expression system as racemic mixture and as single enantiomer. Alkaline liquid/liquid extracts of the samples were analyzed with a pH 2.5 buffer comprising 50 mM Tris and phosphoric acid together with either multiple isomer sulfated β-cyclodextrin (10 mg/mL) or highly sulfated γ-cyclodextrin (2%, w/v). Data obtained in the absence of ketoconazole revealed that the N-demethylation occurred stereoselectively with Michaelis-Menten (incubation of racemic ketamine) and Hill (separate incubation of single enantiomers) kinetics. Data generated in the presence of ketoconazole as the inhibitor could best be fitted to a one-site competitive model and inhibition constants were calculated using the equation of Cheng and Prusoff. No stereoselective difference was observed, but inhibition constants for the incubation of racemic ketamine were found to be larger compared with those obtained with the incubation of single ketamine enantiomers.

Stereoselective determination of drugs and metabolites in body fluids, tissues and microsomal preparations by capillary electrophoresis (2000-2010)

During the past two decades, chiral capillary electrophoresis (CE) emerged as a promising, effective and economic approach for the enantioselective determination of drugs and their metabolites in body fluids, tissues and in vitro preparations. This review discusses the principles and important aspects of CE-based chiral bioassays, provides a survey of the assays developed during the past 10 years and presents an overview of the key achievements encountered in that time period. Applications discussed encompass the pharmacokinetics of drug enantiomers in vivo and in vitro, the elucidation of the stereoselectivity of drug metabolism in vivo and in vitro, and bioanalysis of drug enantiomers of toxicological, forensic and doping interest. Chiral CE was extensively employed for research purposes to investigate the stereoselectivity associated with hydroxylation, dealkylation, carboxylation, sulfoxidation, N-oxidation and ketoreduction of drugs and metabolites. Enantioselective CE played a pivotal role in many biomedical studies, thereby providing new insights into the stereoselective metabolism of drugs in different species which might eventually lead to new strategies for optimization of pharmacotherapy in clinical practice.

Radial extracorporeal shock wave therapy (rESWT) induces new bone formation in vivo: results of an animal study in rabbits

The aim of this study was to investigate if radial extracorporeal shock wave therapy (rESWT) induces new bone formation and to study the time course of ESWT-induced osteogenesis. A total of 4000 impulses of radial shock waves (0.16 mJ/mm²) were applied to one hind leg of 13 New Zealand white rabbits with the contralateral side used for control. Treatment was repeated after 7 days. Fluorochrome sequence labeling of new bone formation was performed by subcutaneous injection of tetracycline, calcein green, alizarin red and calcein blue. Animals were sacrificed 2 weeks (n = 4), 4 weeks (n = 4) and 6 weeks (n = 5) after the first rESWT and bone sections were analyzed by fluorescence microscopy. Deposits of fluorochromes were classified and analyzed for significance with the Fisher exact test. rESWT significantly increased new bone formation at all time points over the 6-week study period. Intensity of ossification reached a peak after 4 weeks and declined at the end of the study. New bone formation was significantly higher and persisted longer at the ventral cortex, which was located in the direction to the shock wave device, compared with the dorsal cortex, emphasizing the dose-dependent process of ESWT-induced osteogenesis. No traumata, such as hemorrhage, periosteal detachment or microfractures, were observed by histologic and radiologic assessment. This is the first study demonstrating low-energy radial shock waves to induce new bone formation in vivo. Based on our results, repetition of ESWT in 6-week intervals can be recommended. Application to bone regions at increased fracture risk (e.g., in osteoporosis) are possible clinical indications.

Evaluation of radial distribution of cartilage degeneration and necessity of pre-contrast measurements using radial dGEMRIC in adults with acetabular dysplasia

Background The purpose of the present study was to investigate the radial distribution patterns of cartilage degeneration in dysplastic hips at different stages of secondary osteoarthritis (OA) by using radial delayed gadolinium-enhanced magnetic resonance imaging of cartilage (dGEMRIC), and to assess whether pre-contrast measurements are necessary. Methods Thirty-five hips in 21 subjects (mean age ± SD, 27.6 ± 10.8 years) with acetabular dysplasia (lateral CE angle < 25°) were studied. Severity of OA was assessed on radiographs using Tönnis grading. Pre- (T1pre) and post-contrast T1 (T1Gd) values were measured at 7 sub-regions on radial reformatted slices acquired from a 3-dimensional (3D) T1 mapping sequence using a 1.5 T MR scanner. Values of radial T1pre, T1Gd and ΔR1 (1/T1Gd - 1/T1pre) of subgroups with different severity of OA were compared to those of the subgroup without OA using nonparametric tests, and bivariate linear Pearson correlations between radial T1Gd and ΔR1 were analyzed for each subgroup. Results Compared to the subgroup without OA, the subgroup with mild OA was observed with a significant decrease in T1Gd in the anterosuperior to superior sub-regions (mean, 476 ~ 507 ms, p = 0.026 ~ 0.042) and a significant increase in ΔR1 in the anterosuperior to superoposterior and posterior sub-regions (mean, 0.93 ~ 1.37 s-1, p = 0.012 ~ 0.042). The subgroup with moderate to severe OA was observed with a significant overall decrease in T1Gd (mean, 404 ~ 452 ms, p = 0.001 ~ 0.020) and an increase in ΔR1 (mean, 1.17 ~1.69 s-1, p = 0.001 ~ 0.020). High correlations were observed between radial T1Gd and ΔR1 for all subgroups (r = −0.869 ~ −0.944, p < 0.001). Conclusions Radial dGEMRIC without pre-contrast measurements is useful for evaluating different patterns of cartilage degeneration in the entire hip joint of patients with hip dysplasia, particularly for those in early stages of secondary OA.

Understanding Electrophoretic Stacking Of In-Capillary Generated Reaction Products

The separation of small molecules by capillary electrophoresis is governed by a complex interplay among several physical effects. Until recently, a systematic understanding of how the influence of all of these effects is observed experimentally has remained unclear. The work presented in this thesis involves the use of transient isotachophoretic stacking (tITP) and computer simulation to improve and better understand an in-capillary chemical assay for creatinine. This assay involves the use of electrophoretically mediated micro-analysis (EMMA) to carry out the Jaffé reaction inside a capillary tube. The primary contribution of this work is the elucidation of the role of the length and concentration of the hydroxide plug used to achieve tITP stacking of the product formed by the in-capillary EMMA/Jaffé method. Computer simulation using SIMUL 5.0 predicts that a 3-4 fold gain in sensitivity can be recognized by timing the tITP stacking event such that the Jaffé product peak is at its maximum height as that peak is electrophoresing past the detection window. Overall, the length of the hydroxide plug alters the timing of the stacking event and lower concentration plugs of hydroxide lead to more rapidly occurring tITP stacking events. Also, the inclusion of intentional tITP stacking in the EMMA/Jaffé method improves the sensitivity of the assay, including creatinine concentrations within the normal biological range. Ultimately, improvement in assay sensitivity can be rationally designed by using the length and concentration of the hydroxide plug to engineer the timing of the tITP stacking event such that stacking occurs as the Jaffé product is passing the detection window.

Determination of free and total valproic acid in human plasma by capillary electrophoresis with contactless conductivity detection

A new approach for the determination of free and total valproic acid in small samples of 140 μL human plasma based on capillary electrophoresis with contactless conductivity detection is proposed. A dispersive liquid-liquid microextraction technique was employed in order to remove biological matrices prior to instrumental analysis. The free valproic acid was determined by isolating free valproic acid from protein-bound valproic acid by ultrafiltration under centrifugation of 100 μL sample. The filtrate was acidified to turn valproic acid into its protonated neutral form and then extracted. The determination of total valproic acid was carried out by acidifying 40 μL untreated plasma to release the protein-bound valproic acid prior to extraction. A solution consisting of 10 mM histidine, 10 mM 3-(N-morpholino)propanesulfonic acid and 10 μM hexadecyltrimethylammonium bromide of pH 6.5 was used as background electrolyte for the electrophoretic separation. The method showed good linearity in the range of 0.4-300 μg/mL with a correlation coefficient of 0.9996. The limit of detection was 0.08 μg/mL, and the reproducibility of the peak area was excellent (RSD=0.7-3.5%, n=3, for the concentration range from 1 to 150 μg/mL). The results for the free and total valproic acid concentration in human plasma were found to be comparable to those obtained with a standard immunoassay. The corresponding correlation coefficients were 0.9847 for free and 0.9521 for total valproic acid.

Determination of creatine and phosphocreatine in muscle biopsy samples by capillary electrophoresis with contactless conductivity detection

A capillary electrophoresis method with contactless conductivity detection was evaluated as a new approach for quantification of creatine and phosphocreatine in human quadriceps femoris biopsy samples. The running buffers employed consisted of 1 M acetic acid at a pH of 2.3 for the determination of creatine and 50 mM 3-(N-morpholino)propanesulfonic acid/30 mM histidine at a pH of 6.4 for the determination of phosphocreatine in the centrifuged muscle extracts. The limits of detection for creatine and phosphocreatine were found to be 2.5 and 1.0 μM, respectively. Creatine and phosphocreatine were determined in six human muscle biopsy samples and the results were found comparable to those of a standard enzymatic assay. The procedures developed for creatine and phosphocreatine also allow the determination of creatinine as well as adenosine diphosphate and adenosine triphosphate.

Good agreement between capillary and venous sampling for lectin pathway proteins

Background: The lectin pathway of complement activation, in particular mannose-binding lectin (MBL), has been extensively investigated over recent years. So far, studies were exclusively based on venous samples. The aim of this study was to investigate whether measurements of lectin pathway proteins obtained by capillary sampling are in agreement with venous samples. Methods: Prospective study including 31 infants that were admitted with suspected early-onset sepsis. Lectin pathway proteins were measured in simultaneously obtained capillary and venous samples. Bland–Altman plots of logarithmized results were constructed, and the mean capillary to venous ratios (ratiocap/ven) were calculated with their 95% confidence intervals (CI). Results: The agreement between capillary and venous sampling was very high for MBL (mean ratiocap/ven, 1.01; 95% CI, 0.85–1.19). Similarly, high agreement was observed for H-ficolin (mean ratiocap/ven, 1.02; 95% CI, 0.72–1.44), MASP-2 (1.04; 0.59–1.84), MASP-3 (0.96; 0.71–1.28), and MAp44 (1.01; 0.82–1.25), while the agreement was moderate for M-ficolin (mean ratiocap/ven, 0.78; 95% CI, 0.27–2.28). Conclusions: The results of this study show an excellent agreement between capillary and venous samples for most lectin pathway proteins. Except for M-ficolin, small volume capillary samples can thus be used when assessing lectin pathway proteins in neonates and young children.

Capillary zone electrophoresis determination of carbohydrate-deficient transferrin using the new CEofix reagents under high-resolution conditions

Capillary zone electrophoresis (CZE) with a dynamic double coating based on the new CEofix reagents is shown to provide high-resolution separations of serum transferrin (Tf) isoforms, a prerequisite for the monitoring of unusual and complex Tf patterns, including those seen with genetic variants and disorders of glycosylation. A 50 microm I.D. fused-silica capillary of 60 cm total length, an applied voltage of 20 kV and a capillary temperature of 30 degrees C results in 15 min CZE runs of high assay precision and thus provides a robust approach for the determination of carbohydrate-deficient transferrin (CDT, sum of asialo-Tf and disialo-Tf in relation to total Tf) in human serum. Except for selected samples of patients with severe liver diseases and sera with high levels of paraproteins, interference-free Tf patterns are detected. Compared with the use of the previous CEofix reagents for CDT under the same instrumental conditions, the resolution between disialo-Tf and trisialo-Tf is significantly higher (1.7 versus 1.4). The CDT levels of reference and patient sera are comparable, suggesting that the new assay can be applied for screening and confirmation analyses. The high-resolution CZE assay represents an attractive alternative to HPLC and can be regarded as a candidate of a reference method for CDT.

Capillary electrophoresis contributions to the hydromorphone metabolism in man

CE-ESI multistage IT-MS (CE-MS(n), n < or = 4) and computer simulation of fragmentation are demonstrated to be effective tools to detect and identify phase I and phase II metabolites of hydromorphone (HMOR) in human urine. Using the same CE conditions as previously developed for the analysis of urinary oxycodone and its metabolites, HMOR and its phase I metabolites produced by N-demethylation, 6-keto-reduction and N-oxidation and phase II conjugates of HMOR and its metabolites formed with glucuronic acid, glucose, and sulfuric acid could be detected in urine samples of a patient that were collected during a pharmacotherapy episode with daily ingestion of 48 mg of HMOR chloride. The CE-MS(n) data obtained with the HMOR standard, synthesized hydromorphol and hydromorphone-N-oxide, and CYP3A4 in vitro produced norhydromorphone were employed to identify the metabolites. This approach led to the identification of previously unknown HMOR metabolites, including HMOR-3O-glucide and various N-oxides, structures for which no standard compounds or mass spectra library data were available. Furthermore, the separation of alpha- and beta-hydromorphol, the stereoisomers of 6-keto-reduced HMOR, was achieved by CE in the presence of the single isomer heptakis(2,3-diacetyl-6-sulfato)-beta-CD. The obtained data indicate that the urinary excretion of alpha-hydromorphol is larger than that of beta-hydromorphol.

Capillary electrophoretic investigation of the enantioselective metabolism of propafenone by human cytochrome P-450 SUPERSOMES: Evidence for atypical kinetics by CYP2D6 and CYP3A4

An enantioselective CE method was used to identify the ability of CYP450 enzymes and their stereoselectivity in catalyzing the transformation of propafenone (PPF) to 5-hydroxy-propafenone (5OH-PPF) and N-despropyl-propafenone (NOR-PPF). Using in vitro incubations with single CYP450 enzymes (SUPERSOMES), 5OH-PPF is shown to be selectively produced by CYP2D6 and N-dealkylation is demonstrated to be mediated by CYP2D6, CYP3A4, CYP1A2, and CYP1A1. For the elucidation of kinetic aspects of the metabolism with CYP2D6 and CYP3A4, incubations with individual PPF enantiomers and racemic PPF were investigated. With the exception of the dealkylation in presence of R-PPF only, which can be described by the Michaelis-Menten model, all CYP2D6-induced reactions were found to follow autoactivation kinetics. For CYP3A4, all NOR-PPF enantiomer formation rates as function of PPF enantiomer concentration were determined to follow substrate inhibition kinetics. The formation of NOR-PPF by the different enzymes is stereoselective and is reduced significantly when racemic PPF is incubated. Clearance values obtained for CYP3A4 dealkylation are stereoselective whereas those of CYP2D6 hydroxylation are not. This paper reports the first investigation of the PPF hydroxylation and dealkylation kinetics by the CYP2D6 enzyme and represents the first report in which enantioselective CE data provide the complete in vitro kinetics of metabolic steps of a drug.