Analytical Approaches for the Quantitation of Redox-active Pyridine Dinucleotides in Biological Matrices

Some of the main electron carriers in the metabolism are mono- or dinucleotides and they play crucial roles in maintaining a balanced redox homeostasis of cells, and in coupling many anabolic and catabolic reactions. Altered cellular redox status can be an indicator of various metabolic disorders such as obesity, the metabolic syndrome, or type 2 diabetes and of other pathological conditions, which involve oxidative stress, such as cardiovascular diseases. Adequate NAD+/NADH and NADP+/NADPH ratios are fundamental for normal cellular functions, thus accurate measurement of these pyridine dinucleotides is essential in biochemical research. Liquid chromatography coupled to tandem mass spectrometry has become the leading analytical technology in (targeted) state-of-the-art metabolic profiling. Main difficulties that hamper quantification of metabolites are chemical similarities, high polarity, and chemical and biological instability of the molecules to be measured. In this review, some critical steps of studying cellular redox status are described, in particular, different techniques of sample preparation and challenges in chromatographic separation

Relationship between Commonly Used Adsorption Isotherm Equations Impedes Isotherm Selection

If the measured isotherm data of an adsorption system are well described by the Freundlich equation, then they can similarly well be described by the bi-Langmuir or tri-Langmuir model in most practical cases. This is proved by Monte Carlo simulation and by comparison of the mathematical functions of the respective isotherm models

Simultaneous Quantitative Determination of Different Ceramide and Diacylglycerol Species in Cultured Cells by Using Liquid Chromatography–Electrospray Tandem Mass Spectrometry

A sensitive, reproducible reverse-phased high performance liquid chromatography electrospray tandem mass spectrometry (HPLC-ESI-MS/MS) method with simple sample preparation was developed for the simultaneous determination of a wide range of ceramides, diacylglycerols (DAGs) in cultured cells. Chromatographic separation of the compounds was achieved in a 14-minute run using a C8 column with a gradient elution by methanol and 10 mM ammonium acetate buffer as mobile phase at a flow rate of 0.5 ml/min. Various ceramides, DAGs were detected with a triple quadrupol system in multiple reaction monitoring mode, which is based on a soft positive electrospray ionization. The usual sample preparation process was shortened by the application of pure methanol for the extraction instead of the widely used methanol/chloroform mixture. C17:0 ceramide which does not occur in the cell samples, was used as an internal standard. The sample preparation process was optimized and the methodology was tested on a human hepatocarcinoma cell culture. Our results clearly showed accumulation of some ceramides and DAGs in the cells treated with BSA-conjugated palmitate for 8 hours. Since both ceramides and DAGs are important lipid intermediates and signal messengers, alteration in their cellular levels have major impact on cell functions, and thus our novel analytic method can be widely used in lipotoxicity research. The presented technique can be further developed to measure other intermediates of ceramide synthesis and other derivatives of DAGs as well

Application of Gas Chromatography – Flame Ionization Detection to Study Cellular Incorporation of Dietary Trans Fatty Acids of Medical Importance

Putative health effects of dietary trans fatty acids (TFAs) receive a growing attention; while very little is known about the metabolism of these special food components. In vitro studies carried out in cultured cells provide an efficient and standardizable approach to follow the metabolic fate of TFAs, but it requires suitable techniques for the quantitative measurement of FAs in cell samples. Here, the development and validation of a simple and reliable method for the quantification of a group of relevant FAs by gas chromatography and flame ionization detection is presented. Sample preparation used a fast one-step and chloroform-free process for simultaneous extraction and esterification, and chromatographic separation was achieved in 25 min using a Zebron ZB-88 capillary column. A linear calibration (of R2 >0.99) was obtained in the concentration range of 1-200 µg/mL for each FA. Recovery rate was 82 % for samples of non-esterified FAs and >95 % for complex lipids, such as ceramides, diglycerides and triglycerides. The LOD and LOQ were below 0.5 µg/mL, and a robust method precision was achieved (RSD % was below 6 % for each lipid classes). The present method was also tested on a cultured cell line with or without FA treatment at close to physiological concentration, and the observed changes in the metabolite concentration levels revealed characteristic differences between the metabolism of cis and trans unsaturated FAs

A novel method of molecular imprinting applied to the template cholesterol

A novel method is successfully tested for non-covalent imprinting. Conditions are used which practically exclude the formation of prepolymerization complexes. The template is cholesterol, and no so-called functional monomer is used. The polymers contain only an acrylic diester crosslinker. The porogen isopropanol prevents even hydrogen bonding between the template and the monomer in the prepolymerization solution. Despite of these apparently very disadvantageous conditions, appreciable imprinting factors for cholesterol and imprinted selectivity against some other steroids are observed, similar to other cholesterol MIPs with proven analytical usefulness