Current Analytical Chemistry - Volume 4, Issue 1, 2008

It is well known that cholesterol is localized in a non-random distribution within and across biological membranes. The importance of the cholesterol-enriched domains, also termed rafts, is evident from the fact that non-receptormediated cholesterol uptake and reverse cholesterol transport also occur through select plasma membrane domains. However, despite much effort to resolve the mechanisms that explain the origin, function, and regulation of membrane cholesterol lateral and transbilayer asymmetric distribution into domains, these phenomena remain largely unresolved. As well, progress in understanding the pathways of intracellular cholesterol trafficking, and the ultimate cellular fate of that cholesterol, has been sparse. Understanding the above-named phenomena and processes is itself crucial to resolving the molecular mechanisms of cholesterol uptake, reverse cholesterol transport, modulation of membrane function, and steroidogenesis. These ongoing efforts to elucidate the nature of cholesterol distribution and dynamics within the cell, have necessitated devising new ways to investigate the trafficking of cholesterol. To that end, fluorescent kinetic exchange assays have been developed to probe the nature of sterol transfer between biological membranes, i.e. endoplasmic reticulum, lysosomes, mitochondria, plasma membrane, and caveolae/lipid rafts (i.e., distinct sub-domains of the plasma membrane). These exchange assays make use of spectroscopic properties, such as polarization, to investigate the nature and distribution of sterol within biological membranes. These assays demonstrate that: cholesterol is distributed within the plane of biomembrane layers into dynamic and static domains, with the latter predominating, and that regulation of the size and kinetics of biomembrane cholesterol domains might be determining factors in intracellular cholesterol trafficking, targeting, and efflux.

Transverse relaxation in the rotating frame (T2ρ) is the dominant relaxation mechanism during a train of adiabatic full passage (AFP) radiofrequency (RF) pulses with no interpulse time intervals placed after the 90° excitation pulse. The magnetization components remain transverse to the time-dependent effective field and undergo relaxation with the time constant T2ρ. Longitudinal relaxation in the rotating frame (T1ρ) is the dominant relaxation mechanism during a train of AFP RF pulses placed prior to an excitation pulse. Here, magnetization is aligned along the time-dependent effective field during adiabatic rotation undergoes relaxation with the time constant T1ρ. A detailed description of rotating frame relaxations due to dipolar interactions and exchange during adiabatic pulses is presented herein. The exchange-induced and dipolar interaction contributions depend on the modulation functions of the adiabatic pulses used. The intrinsic rotating frame relaxation rate constant is sensitive to fluctuations at the effective frequencey (ωeff) in the rotating frame, and this is modulated differently during the two types of AFP pulses. This may lead to the possibility to assess T1ρ and T2ρ relaxation influenced by dipolar relaxation pathways and exchange in human brain tissue and provide a means to generate T1ρ and T2ρ contrasts in MRI.

Characterization of carbohydrate moieties of glycoproteins using microwave-assisted partial acid hydrolysis (MAPAH) and mass spectrometry (MS) are described in this review. Acids including hydrochloric acid (HCl), trifluoroacetic acid (TFA), and phosphoric acid (H3PO4) can be used to induce partial hydrolysis of the carbohydrate moieties of glycoproteins in as short as 30 s of microwave exposure. High resolving power of MS allows distinction between different glycopeptide fragments. Several glycoproteins including bovine pancreatic ribonuclease B (RNase B, MW ∼15 kDa), egg white avidin (MW ∼16 kDa), human serum α1-acid glycoprotein (α1-AGP, MW ∼36 kDa), horseradish peroxidase (HRP, MW ∼44 kDa), fetal calf serum fetuin (BSF, MW ∼45 kDa), and glucose oxidase from Aspergillus niger (GO, MW ∼75 kDa), are used to demonstrate this technique. Information of the sugar composition and/or arrangement of the carbohydrate moieties can be readily obtained either from glycoproteins directly or digested glycopeptides of the glycoproteins. This method is fast, sensitive, and a reliable technique. Challenges when working on larger and heavily glycosylated glycoproteins are discussed.

Longitudinal multispin orders can be created in spin systems that exhibit scalar, dipolar or quadrupolar couplings. They provide an effective way for measurement of scalar couplings and also to probe molecular interactions and dynamics. They cannot be separated by phase cycling or gradient selection methods which are the only known modes of separating different coherences. In this review we describe the frequency cycling procedure for separating various orders in weakly and strongly coupled spin systems. We provide the analytical solutions that permit determination of the frequency cycle for different spin systems. We also discuss the creation of longitudinal orders through relaxation. Finally we highlight the potential applications including spectral editing, measurement of relative signs of scalar couplings and structural properties of molecules.

A review is presented on recent applications of experimental design and optimization techniques for the analysis of compounds of food, biomedical, toxicological and environmental concern. The main features and the significant advantages of chemometric approaches are discussed. Examples related to the determination of substances like xenobiotics or naturally occurring compounds using different analytical techniques, i.e. gas chromatography (GC), liquid chromatography (LC) and capillary electrophoresis (CE) are provided in this Part. The use of experimental design techniques for optimization of extraction techniques is emphasized as well as the importance of experimental design in checking robustness of analytical methods. This survey will attempt to cover the state-of-the-art from 2004 to 2006.