A new electrochemical HPLC method for analysis of enkephalins and endomorphins
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)
- et al.
Enkephalin pseudopetides: resistance to in vitro proteolytic degradation afforded by amide bond replacements extends to remote sites
Peptides
(1985) - et al.
Acupuncture-like stimulation induces a heterosegmental release of met-enkephalin-like material in the rat spinal cord
Pain
(1991) - et al.
Spontaneous and evoked release of met-enkephalin-like material from the spinal cord of arthritic rats in vivo
Pain
(1988) - et al.
Subcutaneous formalin induces a segmental release of met-enkephalin-like material from the rat spinal cord
Pain
(1990) - et al.
Morphine reduces the release of met-enkephalin-like material from the rat spinal cord in vivo by acting at delta opioid receptors
Neuropeptides
(1994) - et al.
Prevention of degradation of enkephalins produces inhibition of nociceptive neurons in rat spinal cord
Brain Res
(1987) - et al.
Grafts of immortalized chromaffin cells bio-engineered to improve met-enkephalin release also reduce formalin evoked c-fos expression in rat spinal cord
Neuorsci Lett
(2004) - et al.
The electron microscopic localization of methionine-enkephalin within the superficial layers (I and II) of the spinal cord
Neuroscience
(1980) - et al.
Comparison of microdialysis and push–pull perfusion for retrieval of serotonin and norepinephrine in the spinal cord dorsal horn
J Neurosci Meth
(2003) - et al.
Release in vivo of Met-enkephalin and encrypted forms of met-enkephalin from brain and spinal cord of the anesthetized cat
Peptides
(1990)
Localization of endomorphin-2-like immunoreactivity in the rat medulla and spinal cord
Peptides
Descending control of pain
Progr Neurobiol
[d-pen2, l-cys5]enkephalinamide and [d-pen2, d-cys5]enkephalinamide, confunctionally constrained cyclic enkephalinamide analogs with delta receptor specificity
Biochem Biophys Res Commun
Analysis of enkephalins, beta-endorphins and small peptides in their sequences by highly sensitive high-performance liquid chromatography with electrochemical detection: implications in opioid peptide metabolism
J Chromatogr
Measurement of proenkephalin A-derived peptides in biological tissues by high pressure liquid chromatography coupled with amperometric electrochemical detection
Life Sci
Release of [Met5]enkephalin from the spinal cord by intraventricularly administered endomorphin-2, but not endomorphin-1 in the anesthetized rat
Neurosci Lett
Immunocytochemical mapping of endomorphin-2-immunoreactivtiy in rat brian
J Chem Neuroanatomy
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