A new electrochemical HPLC method for analysis of enkephalins and endomorphins

https://doi.org/10.1016/j.jneumeth.2005.06.001Get rights and content

Abstract

Endogenous opioid peptides, enkephalins and endomorphins, are located in key regions involved in pain transmission and analgesia, including the spinal cord. These endogenous peptides activate opioid receptors to produce analgesia and reduce pain. We describe a new method to measure enkephalin and endomorphins by high performance liquid chromatography with electrochemical detection. This method allows use of a small sample volume to measure met-enkephalin, leu-enkephalin, endomorphin-1 and endomorphin-2 simultaneously. Using push–pull perfusion of the spinal cord, there were detectable concentrations of met-enkephalin, leu-enkephalin, and endomorphin-2. Further infusion of 100 mM potassium chloride evoked release of met-enkephalin and endomorphin-2 but not leu-enkephalin. Thus, we have developed a method to simultaneously measure enkephalins and endomorphins in small sample volume that allows measurement of these opioid peptides in vivo.

Introduction

Endogenous opioid peptides play a critical role in the body's response to tissue injury. Methionine (met)-enkpehalin, leucine (leu)-enkephalin, and endomorphin-2 are located in key regions involved in pain transmission and analgesia, including the spinal cord (Cheng et al., 1995, Hunt et al., 1980, Martin-Schild et al., 1997, Millan, 2002, Qinyang et al., 2005). Exogenous application endomorphin-1, endomorphin-2, met-enkephalin and leu-enkephalin are antinociceptive, reduce tissue injury hyperalgesia, and dorsal horn neuron activity (Chapman et al., 1997, Dickenson et al., 1987, Millan, 2002, Sakurada et al., 2001, Schmauss and Yaksh, 1984, Wilcockson et al., 1984). Endogenous release of these opioid peptides in the spinal cord has been measured in a number of studies in response to tissue injury, dorsal root stimulation, or stimulation of supraspinal pain inhibition sites (Ballet et al., 2000, Bing et al., 1991, Bourgoin et al., 1988, Bourgoin et al., 1990, Dun et al., 2000, Lucas and Yaksh, 1990, Przewlocka et al., 1992, Suh et al., 1992, Tseng and Huang, 1992, Williams et al., 1999). The majority of studies measure opioid concentrations with radioimmunoassay (RIA) or with enzyme-linked immunoassay (ELISA). Both of these techniques utilize high sample volume (200–500 μl) and are generally less sensitive than electrochemical high performance liquid chromatography (HPLC) (Ballet et al., 2000, Collin et al., 1994, Duplan et al., 2004, Ohsawa et al., 2001, Williams et al., 1999). However, these studies show release of endmorphin-2 and met-enkephalin. To our knowledge there are no reports of evoked release of leu-enkephalin in the spinal cord, or simultaneous measurement of endomorphins or enkephalins. To measure continuous release of several peptides over time with microdialysis or push–pull perfusion, low sample volume (10–20 μl) and increased sensitivity is needed.

HPLC with electrochemical detection is one of the most accurate methods for determining the concentrations of neurotransmitters and allows for the measurement of small samples volumes. Currently, several high performance liquid chromatography (HPLC) methods are available to measure met- and leu-enkephalin that utilize electrochemical detection, fluorescent detection, and ultraviolet detection (Benovitz and Spatola, 1985, Fleming and Reynolds, 1984, Mousa and Couri, 1983, Mousa and Van Loon, 1985). These methods utilize an acidic mobile phase with a reverse phase column. However, there is no HPLC technique for measuring endomorphin-1 or -2. Further, there is no technique available to measure all four peptides in one sample with a small volume. Therefore, we developed an isocratic HPLC method to measure the enkephalins and endomorphins in one sample, with small volume (50 μl), increased precision, and increased sensitivity. The HPLC assay utilizes an acidic mobile phase with a reverse phase column and electrochemical detection. Utilizing push–pull perfusion of the spinal cord with this new electrochemical HPLC method we examined concentrations of met-enkephalin, leu-enkephalin, endomorphin-1 and endomorphin-2 in the spinal cord dorsal horn.

Section snippets

HPLC method

The column was a Prodigy 5μ ODS 100 A, 150 mm × 4.6 mm manufactured by Phenomenex (Torrance, CA) with a corresponding guard column. Prodigy stationary phase is polar endcapped with aromatic and polar compound selectivity. Endcapping offers a surface with almost no silanol activity, meaning the surface of the packing material is inert when compared to normal octadecylsilane columns. In addition to the other benefits, Prodigy is stable under acidic conditions.

The mobile phase was prepared by mixing

Electrochemical HPLC method

To determine the appropriate voltages for reduction and subsequent oxidation of each compound a voltagram was created. To create the voltagram, known starting voltages for leu-enkephalin were used. From the starting voltages we created a voltagram. The reduction voltage for the first electrochemical cell was determined to be anywhere from −200 to −100 mV. Therefore, experiments were done with −150 mV. The optimal oxidation voltage was determined to be approximated +350 mV (Fig. 1).

The method was

Discussion

The current data show that we can detect leu-enkephalin, met-enkephalin, endomorphin-1 and endomorphin-2 in one sample with HPLC coupled to electrochemical detection. This is a sensitive assay, utilizes only 50 μl of sample, and is sufficient to detect the opioid peptides in the extracellular fluid of the spinal cord. The detection limit in fg/ml is 10,000 times more sensitive than extracellular cerebrospinal fluid concentrations in ng/ml. Furthermore, we can measure evoked changes in

Acknowledgements

We thank Ms. Carol Leigh for secretarial assistance. This was supported by the Arthritis Foundation and K02 AR02201.

References (35)

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