Review

High-performance liquid column chromatography of nucleotides, nucleosides and bases

Classical concepts of peripheral neurotransmission were insufficient to explain enteric inhibitory neurotransmission. Geoffrey Burnstock and colleagues developed the idea that ATP or a related purine satisfies the criteria for a neurotransmitter and serves as an enteric inhibitory neurotransmitter in GI muscles. Cloning of purinergic receptors and development of specific drugs and transgenic mice have shown that enteric inhibitory responses depend upon P2Y₁ receptors in post-junctional cells. The post-junctional cells that transduce purinergic neurotransmitters in the GI tract are PDGFRα⁺ cells and not smooth muscle cells (SMCs). PDGFRα⁺ cells express P2Y₁ receptors, are activated by enteric inhibitory nerve stimulation and generate Ca²⁺ oscillations, express small-conductance Ca²⁺-activated K⁺ channels (SK3), and generate outward currents when exposed to P2Y₁ agonists. These properties are consistent with post-junctional purinergic responses, and similar responses and effectors are not functional in SMCs. Refinements in methodologies to measure purines in tissue superfusates, such as high-performance liquid chromatography (HPLC) coupled with etheno-derivatization of purines and fluorescence detection, revealed that multiple purines are released during stimulation of intrinsic nerves. β-NAD⁺ and other purines, better satisfy criteria for the purinergic neurotransmitter than ATP. HPLC has also allowed better detection of purine metabolites, and coupled with isolation of specific types of post-junctional cells, has provided new concepts about deactivation of purine neurotransmitters. In spite of steady progress, many unknowns about purinergic neurotransmission remain and require additional investigation to understand this important regulatory mechanism in GI motility.

In mono-layered primary cell cultures baseline AMP and ADP levels are found nominally in the mid to low picomolar range and are thus difficult to measure with conventional HPLC methods that often require the pooling of samples or require indirect detection methods using radiotracers or enzyme coupled assays. To address this issue, we developed a highly sensitive and selective ion-pairing HPLC method with fluorescence detection to quantify adenine nucleotides and the adenylate energy charge in primary astrocyte cell cultures. To accomplish this, we optimized the fluorescence derivatization conditions and the HPLC parameters to achieve baseline separation and quantification of all adenine nucleotides. Nucleotides were converted to their respective 1, N⁶-etheno derivatives by incubating with chloroacetaldehyde at pH 4.5 and 60 °C for 60 min. Under these conditions, the loss of the adenine nucleotides due to hydrolysis was minimized with a derivatization yield of 94.1% for 1, N⁶-ethenoadenosine. The optimal concentration of tetrabutylammonium phosphate, the ion-pairing reagent, required to achieve a reproducible separation of the adenine nucleotides was found to be 0.8 mM. Calibration curves of nucleotide standards were linear within the range of 0.16–10.4 pmol for adenosine, 0.16–20.6 pmol for AMP, 0.15–19.2 pmol for ADP, and 0.15–19.5 pmol for ATP. The limits of detection and quantification for all adenine nucleotides were approximately 0.08 and 0.16 pmol, respectively. The intra- and inter-day variability for this method was less than 5.1 and 3.4%, respectively. This method was successfully used to measure all adenine nucleotides and an adenylate energy charge of 0.92 ± 0.02 in primary astrocyte cell cultures.

Knowledge of the energetic state of tissue is required in a wide range of experimental studies, particularly those investigating the decline and recovery of cellular metabolism after metabolic stress. Such information can be obtained from high-performance liquid chromatography (HPLC) determination of tissue levels of adenine nucleotides (ATP, ADP, and AMP) and their interrelationship in the tissue energy charge (EC). Accordingly, a large range of techniques with which to measure these molecules and their downstream metabolites have been reported. However, the accurate determination of the tissue EC also depends on the nucleotide extraction procedure given that changes in adenine nucleotide levels take place very quickly when ATPases are not inactivated immediately. In this article, we describe an ion-pair reversed-phase HPLC method by which separation of adenine nucleotides can be performed rapidly, allowing multiple analyses in 1 day, with both high sensitivity and extraction efficiency and using fresh samples, thereby avoiding freeze–thaw degradation of nucleotides. We applied this method to hippocampal brain slice extracts and show that same-day extraction and analysis results in a more accurate determination of the in situ energetic state than does the commonly used snap-freezing in liquid nitrogen.

This chapter discusses approaches to measure adenosine triphosphate (ATP) synthesis from mammalian cells and tissues and presents the procedures to estimate the steady-state content of ATP and other high-energy phosphates by high-performance liquid chromatography (HPLC). The isolation of highly coupled mitochondria from cultured cells involves delicate and time-consuming procedures; the results are sometimes inconsistent, leading to a potential lack of reproducibility. Measurement of ATP synthesis on whole cells requires a permeabilization step to allow for the penetration of hydrophilic substrates through biological membranes. In addition, the use of permeabilized cells rather than isolated mitochondria substantially reduces the number of cells required for each assay. Fluorimetry is a commonly used method to detect ATP produced by isolated mitochondria. Another method employed on isolated mitochondria is the incorporation of Pi into ADP and its subsequent transfer to glucose-6-phosphate by hexokinase, followed by extraction of unincorporated Pi and measurement of radioactivity in a scintillation counter. Among the different methods used to assay high-energy phosphates in biological samples, HPLC has the advantage of high sensitivity and efficiency because it allows for the simultaneous analyses of all species of phosphorylated nucleotides in one analysis. The chapter also describes the assay of ATP synthesis in five cell types—HeLa, COS-7, N2A, HEK 293T, and 143 B-derived cytoplasmic hybrids harboring either wild-type mitochondrial DNA (mtDNA) or the T8993G mtDNA mutation in the ATPase 6 gene, which is responsible for a mitochondrial disorder characterized by neuropathy, ataxia, and retinitis pigmentosa.

Drug efflux pumps like P-glycoprotein (P-gp) and multidrug resistance (MDR) proteins were recognized to posses functional role in determining the pharmacokinetics of drugs administered by peroral as well as parenteral route. Advancements in molecular biology, to some extent, had revealed the structure, localization and functional role of P-glycoprotein and its mechanism of drug efflux. Broad substrate recognization by this protein and clinical implications of its inhibition has revolutionized cancer chemotherapy leading to design and development of novel P-glycoprotein inhibitors. In the recent times, the application of these inhibitors in improving peroral drug delivery has gained special interest. Inhibition of P-glycoprotein improves intestinal absorption and tissue distribution while reducing the substrate metabolism and its elimination. Eventually, various screening methodologies have been developed for determining the activity of P-glycoprotein, kinetics of drug transport and identification of substrates and inhibitors. In the present review, techniques used for screening P-glycoprotein inhibitors and the scope of these inhibitors in optimizing peroral drug absorption and pharmacokinetics are discussed along with a brief introduction to P-glycoprotein, its physiological function and active role in extrusion of drugs.

The purpose of this chapter is to provide information on the fluorescent nucleotides. A proper characterization of fluorescent nucleotides requires reporting of the absorption spectrum and molar extinction coefficient. Molar extinction coefficients are useful because the exact mass of many nucleotide preparations is not known. Fluorescent nucleotides are widely used to study the structure and function of proteins and nucleic acids. With the advent of methods to study macromolecules at the single-molecule level with fluorescence imaging techniques, and the advent of two-photon excitation technologies, the interest in new nucleofide derivatives with appropriate fluorescent properties has increased. Basic information about the general approaches to synthesis such as reagent preparation, monitoring of synthetic reactions, chromatographic methods, spectral characterization, and compositional analysis are provided in the chapter to prepare new compounds. The general classes of nucleotide derivatives are also summarized and general synthetic routes are described in the chapter. Specific protocols are provided only when the synthesis demonstrates a route to a general class of compounds.