Upsala J Med Sci 92: 37-45, 1987 Endorphin Activity in Cerebrospinal Fluid Prior to Elective Cesarean Section and in Early Puerperium Sven Lyrenas,' Fred N ~ b e r g , ~ Gunilla Willdeck-Lund,' Leif L i n d ~ t r o m , ~ Bo Lindberg' and Lars Terenius4 'Departments o Obstetrics and Gynecology and 'Anesthesiology, University Hospital, Uppsala, fPsychiatjic Research Center, and 4Department of Pharmacology, University of Uppsala, Uppsala, Sweden ABSTRACT Opioid activity in cerebrospinal fluid (CSF) was estimated by radioreceptor- assay (RRA) in samples obtained from ten women a t term pregnancy and in the early puerperal period. The samples were fractioned on Sephadex G-10 columns and two opioid receptor active fractions, FI and FII, were recovered. Two pools of FII materials from pregnant and puerperal women, respectively, were further analyzed by electrophoresis and the concentrations of opioid activity were measured by radioreceptorassay. There was a significant rise in receptor-assayed FII opioid activity in late pregnancy a s well a s in the early puerperal period compared to that of healthy, nonpregnant, nonpuerperal females. Pooled FII material obtained before delivery could be separated into two major components tentatively assigned the hexapep- tide [Metlenkephalin-Lys6 and the heptapeptide [Metlenkephalin-Arg6-Phe7. These two opioid peptides both have their origin in the [Metlenkephalin precur- s o r , proenkephalin A . In the puerperal period there was predominance of only one of these components. @ INTRODUCTION The known number of endogenous opioid peptides (endorphins) which may act as ligands to opiate receptors has increased rapidly in the past few years. By the use of molecular genetics, the amino acid sequences of three different pep- tide precursors to these peptides have been predicted. The f i r s t , proopio- melanocortin, is the precursor of p-endorphin ( 1 3 ) . The other two a r e proenkephalin A (1, 6 , 1 2 ) and proenkephalin B ( l o ) , precursors to enkephalins and dynorphins , respectively. Thus, there a r e three families of endorphins 37 which show affinity in varying degree t o different types of opioid receptors. Endogenous opioids have been reported t o provide pain relief in labour. Thus Oyama et al. in 1980 (18) described complete analgesia in 1 4 women who had received 1 mg of p-endorphin by intrathecal injection in the early stage of labour. A gradual rise in the pain threshold for electric stimuli during the final week prior to parturition has also been registered in r a t experiments ( 4 ) . The pain threshold was rapidly normalized following delivery and it was also abolished in naltrexone treated pregnant animals. Prenatal naloxone also affected morphine sensitivity of the off-spring (7). In the present investigation a screening of cerebrospinal fluid (CSF) sub- stances with opioid receptor affinity was conducted in late pregnancy and in the puerperal period. CSF samples were obtained near the end of the third trimester before onset of labour and also during the early puerperal period in the same women. The aim of the study was to establish whether the amounts of endorphins in CSF a r e increased in the final stage of pregnancy and if such an increase could be related to any of the three opioid peptide precursors. MATERIAL AND METHODS The study included two groups of women. 1) One group was composed of 10 healthy pregnant women (mean age 30 years, range 22-37) who underwent elective cesarean section a t term due t o pelvic disproportion. The women had all volunteered for the study. There were no drugs given prior to delivery. The operations were per- formed during spinal anesthesia and prior to administration of the anesthetic d r u g , a 5 ml sample of CSF was removed through a 25 gauge needle. The cesarean sections were all performed between 10 and 1 2 am and the anesthetic used was 50-75 mg of Xylocain@. A second lumbar puncture was done one week post partum when another 5 ml of CSF was sampled. 2) The control group was composed of 16 nonpregnant, nonpuerperal healthy females who volunteered for the s t u d y . The mean age of this group was 26.1 years (range 18-35). Samples of 5 ml CSF were obtained from each woman with the same technique as for the women in group 1 . The investigation was approved by the Ethics Committee of the Medical The lumbar punctures were done at the level of L:3-L:4 with the woman in a Faculty. 38 sitting position. The average collection time was 15 minutes. CSF samples were taken in plastic tubes, frozen and kept a t -7OOC until analyzed. Biochemical methods The standard peptides [Metlenkephalin and [Metlenkephalin-Arg6-Phe7 were obtained from Bachem, Bubendorf , Switzerland, whereas dynorphinl-8 and [Met] enkephalin-Lys6 were supplied by Peninsula Laboratories, San Carlos, USA. Equipment for gel filtration (Sephadex@ G-10) and the agarose (Agarose-C) used in the electrophoretic separation were obtained from Pharmacia Fine Chemicals, Uppsala , Sweden. The opioid activity in each sample was analyzed according to Terenius and Wahlstrom (20). In this method the opioid active material is separated into two major fractions called FI and FII, by chromatography on Sephadex G-10 columns. These fractions a r e further tested for receptor-binding affinity in a radioreceptorassay (RRA) , which uses synaptic plasma membranes from r a t brain excluding cerebellum with 3H-labelled dihydromorphine as a competing radio- ligand. Each assay included a calibration curve with [Metlenkephalin, and the binding activity of the tested fractions was expressed in [Met] enkephalin equivalents. The biochemical characteristics of these fractions have been studied extensively in this laboratory (15, 16, 17). The contents of fraction I include hydrophilic peptides with eight or more amino acids such a s dynorphin A ( d y n ~ r p h i n ~ - ~ ~ ) and its shorter fragments (i. e . dynorphinl-8) while fraction JI contains enkephalin peptides with six to eight amino acids including [Met] enkephalin-Lys6 and [Met] enkephalin-Arg6-Phe7. Column electrophoresis in agarose suspension of pooled FII material was performed according to Nyberg and Terenius (16). For this procedure, the freeze-dried FII materials from the ten pregnant women were combined into two pools, one consisting of material collected in term pregnancy before cesarean section and one consisting of FII material from samples collected in the early puerperal period. The duration of each electrophoresis was five hours a t a current of 10 mA and a voltage of 1,000 V. Fractions of 0 . 4 ml were then suctioned from the column and separated from agarose by the use of a centri- fuge. The distribution of the endorphin activity was monitored by radioreceptor- assay. Calibration of the column had been performed earlier with samples of known enkephalin- and dynorphin-related peptides . 39 RESULTS The CSF levels of FI and FII in the cesarean section group and in the control group a r e shown in Figs. 1 and 2 . In these patients, no general in- crease was found in FI either a t the time of cesarean section or in the early puerperium in comparison with the control group. F I I , on the other hand, was significantly increased both before cesarean section and in the early puerperium compared to the control group (pC0.01 and p