Antinociceptive effect of intrathecal microencapsulated human pheochromocytoma cell in a rat model of bone cancer pain.

Human pheochromocytoma cells, which are demonstrated to contain and release met-enkephalin and norepinephrine, may be a promising resource for cell therapy in cancer-induced intractable pain. Intrathecal injection of alginate-poly (l) lysine-alginate (APA) microencapsulated human pheochromocytoma cells leads to antinociceptive effect in a rat model of bone cancer pain, and this effect was blocked by opioid antagonist naloxone and alpha 2-adrenergic antagonist rauwolscine. Neurochemical changes of cerebrospinal fluid are in accordance with the analgesic responses. Taken together, these data support that human pheochromocytoma cell implant-induced antinociception was mediated by met-enkephalin and norepinephrine secreted from the cell implants and acting at spinal receptors. Spinal implantation of microencapsulated human pheochromocytoma cells may provide an alternative approach for the therapy of chronic intractable pain.

Preparation of a pipette tip-based molecularly imprinted solid-phase microextraction monolith by epitope approach and its application for determination of enkephalins in human cerebrospinal fluid.

In this study, a novel molecularly imprinted polymer (MIP) monolith for highly selective extraction of enkephalins was synthesized and prepared in a micropipette tip using epitope imprinting technique. The synthesized MIPs were characterized by scanning electron microscope (SEM) and infrared spectroscopy. A molecularly imprinted solid-phase microextraction (MISPME) method was developed for extraction of enkephalins in aqueous solutions. The parameters affecting MISPME were optimized. The results indicated that this MIP monolith exhibited specific recognition capability, high enrichment efficiency and excellent reusability for enkephalins. MALDI-TOF MS analysis demonstrated that this MIP monolith can act as a useful tool for highly selective purification and enrichment of enkephalin, a kind of low abundance protein, from high-abundance proteins in human cerebrospinal fluids (CSF). Employed this MIP monolith as solid-phase microextraction column, quantitative assay of enkephalins in human CSF was developed by HPLC-ultraviolet (UV) detection in this work. The detection limits were 0.05-0.08nM. This MISPME/HPLC-UV method was used to quantify Met-enkephalin and Leu-enkephalin levels in the CSF of patients with cancer pain.

Increased levels of Met-enkephalin-like material in the CSF of anaesthetized cats after tooth pulp stimulation.

Tooth pulp stimulation in halothane-anaesthetized cats induced a long lasting (greater than or equal to 3 h) increase in the levels of Met-enkephalin-like material (MELM) in the cisternal CSF. Chromatographic analyses (gel filtration, HPLC) revealed that most of the immunoreactivity was attributable to high molecular weight (mol. wt. greater than or equal to 4000) compounds; in non-stimulated cats, Met-enkephalin (largely in the form of the sulfoxide derivative) only accounted for about 10% of total MELM. In contrast, following tooth pulp stimulation, a large increase in Met-enkephalin (plus Met-Ox5-enkephalin) levels was noted so that the pentapeptide thus represented more than 50% of total MELM. No evidence was obtained for the presence of Met-enkephalin-Arg6-Phe7 in the cisternal CSF of halothane-anaesthetized cats. These data strongly suggest that the activity of enkephalinergic neurons was increased following nociceptive stimulation. This indirectly supports the possible physiological role of enkephalinergic systems in modulating nociceptive inputs.

Measurement of enkephalin peptides in canine brain regions, teeth, and cerebrospinal fluid with high-performance liquid chromatography and mass spectrometry.

Endogenous enkephalin pentapeptides are measured with unambiguous molecular specificity in canine and human tissue and fluid extracts. Both field desorption and fast atom bombardment mass spectrometry have been used to produce a protonated molecular ion of the peptide high-performance liquid chromatography fraction. The protonated molecular ion is subjected to collision-activated dissociation processes and a linked-field scan (B/E) selects a unique amino acid sequence-determining ion for monitoring and measurement. Stable isotope-incorporated peptide internal standards are used for quantification. Endogenous enkephalins are measured in hypothalamus, cerebrospinal fluid, pituitary, caudate nucleus, and tooth pulp extracts. Part-per-billion levels of endogenous peptide are measured.