Studies on the level of substance P and calcitonin gene-related peptide in cerebrospinal fluid of pregnant women and patients with chronic pain and in rat brain after spinal cord injury : Special reference to analytical aspects of neuropeptides

Abstract: Substance P (SP) and calcitonin gene-related peptide (CGRP) are sensory peptides known to be involved innociception and inflammatory processes. It is well demonstrated that the two peptides can be released from spinal cord neurons during inflammation and chronic pain induced by peripheral nerve injury. Analyses of neuropeptides in the cerebrospinal fluid and in central nervous system tissues are of importance not only for basic research but also for diagnostic purposes.In the present thesis, studies have been directed on the CSF levels of SP and CGRP in pregnant womenand patients with chronic pain (fibromyalgia, osteoarthritis of hip and knee, herniated lumbar disc).Studies on a SP specific endopeptidase (SPE) present in CSF is also included. The level of the peptides was assessed by a pre-extraction procedure combined with radioimmunoassay (RIA), whereas the SPE activity was detected by following the conversion of SP to its heptapeptide fragment SP1-7, using a specific RIA for the product. The identity of the peptide immunoreactivitry was confirmed using high performance liquid chromatography. The procedure outlined for pm-extraction or pm-separation of the CSF samples was based on liquid-liquid extraction. In women at term pregnancy the CSF level of SP was significantly increased in contrast to the CSF activity of SPE, which displayed a significant decrease. The SP levels in CSF from patients with fibromyalgia and osteoarthritis were significantly elevated but remained unchanged in patients with herniated lumbar disc. The CSF level of CGRP was found to be significantly reduced in patients with fibromyalgia. Studies on patients with fibromyalgia syndrome (FMS) also included assessment of the level of the opioid peptide Met-enkephalin-Arg-Phe (MEAP). It was found that the level of CSF MEAP was reduced in FMS patients.The liquid-liquid extraction (LLE) method developed for pretreatment of CSF samples before RIA is amodification of a procedure earlier used for determination of peptides in tissue samples. Factors that may affect the recovery of neuropeptides from CSF were subjected to particular studies. It was found that the protein content in CSF is a major factor influencing the peptide recovery. Acidification of the CSF specimens combined with HC1 was found to be effective to remove proteins in the samples and thereby improve the peptide recovery.Previous studies have shown that CGRP is a potent inhibitor SPE. Therefore, studies on the inhibitoryeffect of CGRP on SP metabolism in both crude human CSF (hCSF) and in a partially purified enzyme-containing CSF fraction were performed. For the first time it was demonstrated that CGRP may inhibit themetabolism of SP1-7, a major metabolic product of SP, in both crude hCSF and the partially purified enzyme fraction. The effects of three protease inhibitors (Amastatin, captopril and phosphoramidon) on SP and SP1-7 metabolism were also investigated using crude hCSF and the partially purified enzyme fraction. Captopril, a potent inhibitor of angiotensin converting enzyme (ACE), was found to inhibit the degradation of SP1-7 in both crude hCSF and by the partially purified enzyme fraction, suggesting that ACE may have a role in the in vivo metabolism of this SP fragment.Spinal cord injury (SCI) was earlier shown to affect the SP levels in the spinal cord at the site of injury but also in adjacent segments. Studies presented in this thesis demonstrates that following SCI to the male rat the undecapeptide is affected also in discrete areas of the rat brain.