Applications of chromatographic methods in metabolomics: A review.

Chromatography is a robust and reliable separation method that can use various stationary phases to separate complex mixtures commonly seen in metabolomics. This review examines the types of chromatography and stationary phases that have been used in targeted or untargeted metabolomics with methods such as mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy. General considerations for sample pretreatment and separations in metabolomics are considered, along with the various supports and separation formats for chromatography that have been used in such work. The types of liquid chromatography (LC) that have been most extensively used in metabolomics will be examined, such as reversed-phase liquid chromatography and hydrophilic liquid interaction chromatography. In addition, other forms of LC that have been used in more limited applications for metabolomics (e.g., ion-exchange, size-exclusion, and affinity methods) will be discussed to illustrate how these techniques may be utilized for new and future research in this field. Multidimensional LC methods are also discussed, as well as the use of gas chromatography and supercritical fluid chromatography in metabolomics. In addition, the roles of chromatography in NMR- vs. MS-based metabolomics are considered. Applications are given within the field of metabolomics for each type of chromatography, along with potential advantages or limitations of these separation methods.

Characterization of binding by sulfonylureas with normal or modified human serum albumin using affinity microcolumns prepared by entrapment.

Modification of proteins can occur during diabetes due to the formation of advanced glycation end-products (AGEs) with reactive dicarbonyls such as glyoxal (Go) and methylglyoxal (MGo). Human serum albumin (HSA) is a serum protein that binds to many drugs in blood and that is known to be modified by Go and MGo. This study examined the binding of various sulfonylurea drugs with these modified forms of HSA by using high-performance affinity microcolumns prepared by non-covalent protein entrapment. Zonal elution experiments were employed to compare the retention and overall binding constants for the drugs with Go- or MGo-modified HSA vs normal HSA. The results were compared to values from the literature, such as measured or estimated using affinity columns containing covalently immobilized HSA or biospecifically-adsorbed HSA. The entrapment-based approach provided estimates of global affinity constants within 3-5 min for most of the tested drugs and with typical precisions of ±10-23%. Each entrapped protein microcolumn was stable for over at least 60-70 injections and one month of use. The results obtained with normal HSA agreed at the 95% confidence level with global affinity constants that have been reported for the given drugs in the literature. It was found for HSA that had been modified with clinically-relevant levels of either Go or MGo that an increase in the global affinity constant of up to 2.1-fold occurred for some of the tested drugs. The information acquired in this study can be used in the future to adapt this entrapment-based approach to study and evaluate interactions between other types of drugs and normal or modified binding agents for clinical testing and biomedical research.

Characterization of binding by repaglinide and nateglinide with glycated human serum albumin using high-performance affinity microcolumns.

High-performance affinity microcolumns were used to characterize binding by the anti-diabetic drugs repaglinide and nateglinide with normal and glycated forms of human serum albumin. The microcolumns contained only nmol amounts of protein and provided a detailed analysis of these drug interactions with good precision and in a matter of minutes per experiment. The overall binding by repaglinide to normal and glycated albumin fits a model with two types of binding sites: a set of one or two moderate-to-high affinity regions and a larger set of weaker regions with association equilibrium constants of ∼105 and 103  M-1 , respectively, at pH 7.4 and 37°C. Competition studies gave site-specific association constants for repaglinide and nateglinide at Sudlow site I of 4.2 × 104 and 5.0 × 104  M-1 for normal albumin, with a decrease of 26%-30% being seen for nateglinide with glycated albumin and no significant change being noted for repaglinide. At Sudlow site II, repaglinide and nateglinide had association constants for normal albumin of 6.1 × 104 and 7.1 × 105  M-1 , with glycated albumin giving an increase in the association constant at this site for repaglinide of 1.6- to 1.8-fold and a decrease for nateglinide of 51%-58%.

High-Performance affinity chromatographic studies of repaglinide and nateglinide interactions with normal and glyoxal- or methylglyoxal-modified human albumin serum.

During diabetes human serum albumin (HSA), an important drug transport protein, can be modified by agents such as glyoxal (Go) and methylglyoxal (MGo) to form advanced glycation end-products. High-performance affinity microcolumns and zonal elution competition studies were used to compare interactions by the anti-diabetic drugs repaglinide and nateglinide with normal and Go- or MGo-modified HSA at Sudlow sites I and II of this protein. Both drugs had their strongest binding at Sudlow site II for the normal and modified forms of HSA. The association equilibrium constants at this site for repaglinide and nateglinide with normal HSA were 6.1 (± 0.2) × 104 M-1 and 7.1 (± 0.8) × 105 M-1, respectively, at pH 7.4 and 37°C; these values increased by up to 3.6-fold for repaglinide and decreased by up to 45-55 % for nateglinide when HSA was modified by Go or MGo at levels seen in prediabetes or diabetes. Both drugs were also found to bind at Sudlow site I, with association equilibrium constants at this site on normal HSA of 4.2 (± 0.3) × 104 M-1 for repaglinide and 5.0 (± 0.1) × 104 M-1 for nateglinide. The binding strength for repaglinide at Sudlow site I increased by 1.3- to 1.7-fold with the Go-modified HSA and decreased slightly (i.e., up to 19 %) for the MGo-modified HSA, while nateglinide showed only a small or insignificant change in binding with the same modified HSA samples. These results indicated that binding by repaglinide and nateglinide with HSA can be altered significantly by modification of this protein with Go or MGo, making these modifications of potential interest in the treatment of patients with these drugs during diabetes.

Kinetic Analysis by Affinity Chromatography.

Important information on chemical processes in living systems can be obtained by the rates at which these biological interactions occur. This review will discuss several techniques based on traditional and high-performance affinity chromatography that may be used to examine the kinetics of biological reactions. These methods include band-broadening measurements, techniques for peak fitting, split-peak analysis, peak decay studies, and ultrafast affinity extraction. The general principles and theory of each method, as applied to the determination of rate constants, will be discussed. The applications of each approach, along with its advantages and limitations, will also be considered.