Characterization of drug binding with alpha1-acid glycoprotein in clinical samples using ultrafast affinity extraction.

Many drugs bind to serum transport proteins, which can affect both drug distribution and activity in the body. α1-Acid glycoprotein (AGP) is a key transport protein for basic and neutral drugs. Both elevated levels and altered glycosylation patterns of AGP have been seen in clinical conditions such as systemic lupus erythematosus (SLE). This study developed, optimized, and used the method of ultrafast affinity extraction (UAE) to examine whether these changes in AGP are associated with changes in the binding by some drugs to this transport protein. This approach used affinity microcolumns to capture and measure, in serum, the free fractions of several drugs known to bind AGP. These measurements were made with pooled normal control serum and serum samples from individuals with SLE. Immunoaffinity chromatography was used to obtain the content of AGP and HSA in these samples, and CE was used to examine the glycoform pattern for AGP in each serum sample. The free drug fractions measured for normal control serum ranged from 3.5 to 29.1%, in agreement with the results of ultrafiltration, and provided binding constants of ~105-106 M-1 for the given drugs with AGP at 37°C. Analysis of a screening set of SLE serum samples by UAE gave decreased free fractions (relative change, 12-55%) vs normal serum when spiked with the same types and amounts of drugs. These changes were related in some cases to AGP content, with some SLE samples having AGP levels 1.3- to 2.1-fold above the upper end of the normal range. In other cases, the changes in free fractions appeared to be linked to alterations in the glycoforms and binding constants of AGP, with some affinities differing by 1.2- to 1.5-fold vs normal AGP. This approach can be employed with other solute-protein systems and to investigate binding by other drugs or transport proteins directly in clinical samples.

Testosterone meets albumin - the molecular mechanism of sex hormone transport by serum albumins.

Serum albumin is the most abundant protein in mammalian blood plasma and is responsible for the transport of metals, drugs, and various metabolites, including hormones. We report the first albumin structure in complex with testosterone, the primary male sex hormone. Testosterone is bound in two sites, neither of which overlaps with the previously suggested Sudlow site I. We determined the binding constant of testosterone to equine and human albumins by two different methods: tryptophan fluorescence quenching and ultrafast affinity extraction. The binding studies and similarities between residues comprising the binding sites on serum albumins suggest that testosterone binds to the same sites on both proteins. Our comparative analysis of albumin complexes with hormones, drugs, and other biologically relevant compounds strongly suggests interference between a number of compounds present in blood and testosterone transport by serum albumin. We discuss a possible link between our findings and some phenomena observed in human patients, such as low testosterone levels in diabetic patients.

Characterization of solution-phase drug-protein interactions by ultrafast affinity extraction.

A number of tools based on high-performance affinity separations have been developed for studying drug-protein interactions. An example of one recent approach is ultrafast affinity extraction. This method has been employed to examine the free (or non-bound) fractions of drugs and other solutes in simple or complex samples that contain soluble binding agents. These free fractions have also been used to determine the binding constants and rate constants for the interactions of drugs with these soluble agents. This report describes the general principles of ultrafast affinity extraction and the experimental conditions under which it can be used to characterize such interactions. This method will be illustrated by utilizing data that have been obtained when using this approach to measure the binding and dissociation of various drugs with the serum transport proteins human serum albumin and alpha1-acid glycoprotein. A number of practical factors will be discussed that should be considered in the design and optimization of this approach for use with single-column or multi-column systems. Techniques will also be described for analyzing the resulting data for the determination of free fractions, rate constants and binding constants. In addition, the extension of this method to complex samples, such as clinical specimens, will be considered.

Nanomaterials as stationary phases and supports in liquid chromatography.

The development of various nanomaterials over the last few decades has led to many applications for these materials in liquid chromatography (LC). This review will look at the types of nanomaterials that have been incorporated into LC systems and the applications that have been explored for such systems. A number of carbon-based nanomaterials and inorganic nanomaterials have been considered for use in LC, ranging from carbon nanotubes, fullerenes and nanodiamonds to metal nanoparticles and nanostructures based on silica, alumina, zirconia and titanium dioxide. Many ways have been described for incorporating these nanomaterials into LC systems. These methods have included covalent immobilization, adsorption, entrapment, and the synthesis or direct development of nanomaterials as part of a chromatographic support. Nanomaterials have been used in many types of LC. These applications have included the reversed-phase, normal-phase, ion-exchange, and affinity modes of LC, as well as related methods such as chiral separations, ion-pair chromatography and hydrophilic interaction liquid chromatography. Both small and large analytes (e.g., dyes, drugs, amino acids, peptides and proteins) have been used to evaluate possible applications for these nanomaterial-based methods. The use of nanomaterials in columns, capillaries and planar chromatography has been considered as part of these efforts. Potential advantages of nanomaterials in these applications have included their good chemical and physical stabilities, the variety of interactions many nanomaterials can have with analytes, and their unique retention properties in some separation formats.

High-Performance Affinity Chromatography: Applications in Drug-Protein Binding Studies and Personalized Medicine.

The binding of drugs with proteins and other agents in serum is of interest in personalized medicine because this process can affect the dosage and action of drugs. The extent of this binding may also vary with a given disease state. These interactions may involve serum proteins, such as human serum albumin or α1-acid glycoprotein, or other agents, such as lipoproteins. High-performance affinity chromatography (HPAC) is a tool that has received increasing interest as a means for studying these interactions. This review discusses the general principles of HPAC and the various approaches that have been used in this technique to examine drug-protein binding and in work related to personalized medicine. These approaches include frontal analysis and zonal elution, as well as peak decay analysis, ultrafast affinity extraction, and chromatographic immunoassays. The operation of each method is described and examples of applications for these techniques are provided. The type of information that can be obtained by these methods is also discussed, as related to the analysis of drug-protein binding and the study of clinical or pharmaceutical samples.