Determination of ethyl glucuronide in human serum by capillary zone electrophoresis and an immunoassay

Ethyl glucuronide (EtG) is a marker of recent alcohol consumption. For the optimization of the analysis of EtG by CZE with indirect absorbance detection, the use of capillaries with permanent and dynamic wall coatings, the composition of the BGE, and various sample preparation procedures, including dilution with water, ultrafiltration, protein precipitation, and SPE, were investigated. Two validated screening assays for the determination of EtG in human serum, a CZE-based approach and an enzyme immunoassay (EIA), are described. The CZE assay uses a coated capillary, 2,4-dimethylglutaric acid as an internal standard, and a pH 4.65 BGE comprising 9 mM nicotinic acid, epsilon-aminocaproic acid and 10% v/v ACN. Proteins are removed via precipitation with ACN prior to analysis and the LOQ is 0.50 mg/L. The EIA is based upon commercial reagents which are promoted for the determination of urinary EtG. Krebs-Ringer solution containing 5% BSA is used as a calibration matrix. All samples are ultrafiltered prior to analysis of the ultrafiltrate on a Mira Plus analyzer. Assay calibration ranged between 0 and 2 mg/L and the upper reference limit was determined to be 0.05 mg/L. Both assays proved to be suitable for the analysis of samples from different individuals. For EtG levels above 0.50 mg/L, good agreement was observed for the comparison of the results of the two methods.

Novel functional profiling approach combining reverse phase protein microarrays and human 3-D ex vivo tissue cultures: expression of apoptosis-related proteins in human colon cancer

Cancer is caused by a complex pattern of molecular perturbations. To understand the biology of cancer, it is thus important to look at the activation state of key proteins and signaling networks. The limited amount of available sample material from patients and the complexity of protein expression patterns make the use of traditional protein analysis methods particularly difficult. In addition, the only approach that is currently available for performing functional studies is the use of serial biopsies, which is limited by ethical constraints and patient acceptance. The goal of this work was to establish a 3-D ex vivo culture technique in combination with reverse-phase protein microarrays (RPPM) as a novel experimental tool for use in cancer research. The RPPM platform allows the parallel profiling of large numbers of protein analytes to determine their relative abundance and activation level. Cancer tissue and the respective corresponding normal tissue controls from patients with colorectal cancer were cultured ex vivo. At various time points, the cultured samples were processed into lysates and analyzed on RPPM to assess the expression of carcinoembryonic antigen (CEA) and 24 proteins involved in the regulation of apoptosis. The methodology displayed good robustness and low system noise. As a proof of concept, CEA expression was significantly higher in tumor compared with normal tissue (p<0.0001). The caspase 9 expression signal was lower in tumor tissue than in normal tissue (p<0.001). Cleaved Caspase 8 (p=0.014), Bad (p=0.007), Bim (p=0.007), p73 (p=0.005), PARP (p<0.001), and cleaved PARP (p=0.007) were differentially expressed in normal liver and normal colon tissue. We demonstrate here the feasibility of using RPPM technology with 3-D ex vivo cultured samples. This approach is useful for investigating complex patterns of protein expression and modification over time. It should allow functional proteomics in patient samples with various applications such as pharmacodynamic analyses in drug development.

Mass spectrometry-based analytical tools for the molecular protein characterization of human plasma lipoproteins

Lipoproteins are a heterogeneous population of blood plasma particles composed of apolipoproteins and lipids. Lipoproteins transport exogenous and endogenous triglycerides and cholesterol from sites of absorption and formation to sites of storage and usage. Three major classes of lipoproteins are distinguished according to their density: high-density (HDL), low-density (LDL) and very low-density lipoproteins (VLDL). While HDLs contain mainly apolipoproteins of lower molecular weight, the two other classes contain apolipoprotein B and apolipoprotein (a) together with triglycerides and cholesterol. HDL concentrations were found to be inversely related to coronary heart disease and LDL/VLDL concentrations directly related. Although many studies have been published in this area, few have concentrated on the exact protein composition of lipoprotein particles. Lipoproteins were separated by density gradient ultracentrifugation into different subclasses. Native gel electrophoresis revealed different gel migration behaviour of the particles, with less dense particles having higher apparent hydrodynamic radii than denser particles. Apolipoprotein composition profiles were measured by matrix-assisted laser desorption/ionization-mass spectrometry on a macromizer instrument, equipped with the recently introduced cryodetector technology, and revealed differences in apolipoprotein composition between HDL subclasses. By combining these profiles with protein identifications from native and denaturing polyacrylamide gels by liquid chromatography-tandem mass spectrometry, we characterized comprehensively the exact protein composition of different lipoprotein particles. We concluded that the differential display of protein weight information acquired by macromizer mass spectrometry is an excellent tool for revealing structural variations of different lipoprotein particles, and hence the foundation is laid for the screening of cardiovascular disease risk factors associated with lipoproteins.

Class II treatment by extraction of maxillary first molars or Herbst appliance: dentoskeletal and soft tissue effects in comparison

AIM To compare dentoskeletal and soft tissue treatment effects of two alternative Class II division 1 treatment modalities (maxillary first permanent molar extraction versus Herbst appliance). METHODS One-hundred-fifty-four Class II division 1 patients that had either been treated with extractions of the upper first molars and a lightwire multibracket (MB) appliance (n = 79; 38 girls, 41 boys) or non-extraction by means of a Herbst-MB appliance (n = 75; 35 girls, 40 boys). The groups were matched on age and sex. The average age at the start of treatment was 12.7 years for the extraction and for 13.0 years for the Herbst group. Pretreatment (T1) and posttreatment (T2) lateral cephalograms were retrospectively analyzed using a standard cephalometric analysis and the sagittal occlusal analysis according to Pancherz. RESULTS The SNA decrease was 1.10° (p = 0.001) more pronounced in the extraction group, the SNB angle increased 1.49° more in the Herbst group (p = 0.000). In the extraction group, a decrease in SNB angle (0.49°) was observed. The soft tissue profile convexity (N-Sn-Pog) decreased in both groups, which was 0.78° more (n. s.) pronounced in the Herbst group. The nasolabial angle increased significantly more (+ 2.33°, p = 0.025) in the extraction group. The mechanism of overjet correction in the extraction group was predominantly dental (65% dental and 35% skeletal changes), while in the Herbst group it was predominantly skeletal (58% skeletal and 42% dental changes) in origin. CONCLUSION Both treatment methods were successful and led to a correction of the Class II division 1 malocclusion. Whereas for upper first molar extraction treatment more dental and maxillary effects can be expected, in case of Herbst treatment skeletal and mandibular effects prevail.

A benchmark of kriging-based infill criteria for noisy optimization

Responses of many real-world problems can only be evaluated perturbed by noise. In order to make an efficient optimization of these problems possible, intelligent optimization strategies successfully coping with noisy evaluations are required. In this article, a comprehensive review of existing kriging-based methods for the optimization of noisy functions is provided. In summary, ten methods for choosing the sequential samples are described using a unified formalism. They are compared on analytical benchmark problems, whereby the usual assumption of homoscedastic Gaussian noise made in the underlying models is meet. Different problem configurations (noise level, maximum number of observations, initial number of observations) and setups (covariance functions, budget, initial sample size) are considered. It is found that the choices of the initial sample size and the covariance function are not critical. The choice of the method, however, can result in significant differences in the performance. In particular, the three most intuitive criteria are found as poor alternatives. Although no criterion is found consistently more efficient than the others, two specialized methods appear more robust on average.

An MEKC assay for the therapeutic drug monitoring of cefepime

The development of a robust assay based on MEKC for cefepime in human serum and plasma with internal quality assurance is reported. Sample preparation comprises protein precipitation in the presence of SDS at pH 4.5. This is a gentle approach for which decomposition of cefepime during sample handling is negligible. After hydrodynamic sample injection of the supernatant, analysis occurs in a phosphate/borate buffer at pH 9.1 with 75 mM SDS using normal polarity and analyte detection at 257 nm. The MEKC run time interval and throughput are about 5 min and seven samples per hour, respectively. The calibration range for cefepime is 1-60 μg/mL, with 1 μg/mL being the LOQ. The performance of the assay with multilevel internal calibration was assessed with calibration and control samples. The assay is shown to be simple, inexpensive, reproducible, and robust. It was applied to determine cefepime levels in the sera of critically ill patients and to assess the instability of cefepime in patient and control samples. Our data revealed that serum containing cefepime can be stored at -20°C for a short time, whereas for long-term storage, samples have to be kept at -70°C.

Informationssuche zwischen Energiepolitik und Energiespartipps. Einstellungs-Verhaltens-Relationen als Erklärungsfaktoren der aktiven Suche nach energiebezogenen Informationen

Im Beitrag wird ein Erklärungsansatz für die aktive Suche nach Informationen zu den Themen Energie, Energieverbrauch und Energieeinsparmöglichkeiten entwickelt und geprüft. Im Mittelpunkt steht dabei die Frage, ob die Diskrepanz zwischen themenbezogenen Einstellungen und Verhaltensweisen einen Einfluss auf die Informationssuche hat. Dazu werden zunächst die Dimensionen dargelegt, die das Themenfeld umfasst und verdeutlicht, warum diese Aspekte von Bedeutung sind. Anschließend werden verschiedene theoretische Ansätze vorgestellt, aus denen sich Einflussfaktoren für die aktive Informationssuche ableiten lassen. Danach wird ein zentraler Befund der sozialwissenschaftlichen Umweltforschung – die häufig zu beobachtende Diskrepanz zwischen Umwelteinstellungen und Umweltverhalten – erläutert und mit der Theorie der kognitiven Dissonanz und dem Konzept der selektiven Zuwendung in Verbindung gebracht. Darauf aufbauend wird ein theoretisches Erklärungsmodell für die Suche nach energiebezogenen Informationen entwickelt. Dieses Modell wird anhand von Paneldaten aus den Jahren 2010 und 2011 empirisch geprüft. Die Befunde zeigen, dass dissonante/konsonante Einstellungs-Verhaltens-Relationen einen Beitrag zur Erklärung der aktiven Informationssuche leisten können, insbesondere dann, wenn Energie für die Personen ein wichtiges Thema ist.