Neuropeptides 4: 217-225, 1984

NORMAL CSF LEVELS OF MET-ENKEPHALIN-LIKE MATERIAL IN A CASE OF NALOXONE-REVERSIBLE CONGENITAL INSENSITIVITY TO PAIN

F. Cesselin, S. Bourgoin, M. Hamon, F. Artaud, M.F. Testut*, A. Rascal* and J.L. Montastruc*

Groupe NB, INSERM LJ.114, College de France, 75005 Paris and *Services de Neurologie et de Pharmacologic MBdicale et Clinique, CHLJ Purpan, Facult6 de MBdecine, Toulouse,France

(reprint requests to F.C.)

ABSTRACT

In a case of naloxone-reversible congenital insensitivity to pain, met-enkephalin-like immunoreactivity in the CSF was in the normal range and not affected by the administration of naloxone. Chromatographic analysis of the met-enkephalin-like material revealed that it corresponded to at least two classes of molecules. A clear difference in the relative proportions of these two classes was detected in the CSF of the patient insensi- tive to pain when compared to controls. The possible functional significance of this alteration is discussed in relation to the well known antinociceptive action of enkephalins.

INTRODUCTION

Considerable evidence supports that endomorphins are involved in the control of nociceptive messages in various species including man. Thus, endomorphin levels rise in the cerebrospinal fluid (CSF) after treatments producing pain relief such as intra- cerebral electrical stimulation (1) and electro- or manual acupuncture (2,3). On the contrary, reduced endomorphin levels have been found in the CSF of chronic (4) or acute (5) pain patients. In addition, the blockade of opiate receptors by naloxone has been claimed to reduce the nociceptive threshold in some (but not all, 6) control subjects (7) and in patients with congenital insensitivity to pain (8). Accordingly, a hyper- activity of some pain-controlling endomorphin systems might be suspected in such patients. The present study was undertaken to verify this hypothesis enkephalin-like material

by measuring the CSF levels of met- (MELM) in one of these patients.

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EXPERIMENTAL PROCEDURES

Case history

The patient, a 26 year-old woman, fourth child of a family with no neuropatholoqical antecedents, presented idiopathic epilepsia (generalized attacks and psychomotor automatisms) which had been well controlled with phenobarbital (150mq/day), phenytoin (200mq/day), clonazepam (2mq/day) and diazepam (20mq/day) since the age of eleven. During a neurological consultation (June 1980), she mentioned feeling no particular pain when she cut herself or was injured. However, she had suffered from nephritic colic pains in 1973 and was subject to premenstrual syndrome symptomatoloqy with the feeling of weight in the pelvis, described as bearable.

On examination, the absence of pain perception was observed over the whole skin. Moreover, cutaneous nociceptive stimulation, whatever its intensity or location, did not provoke the expected withdrawal reaction or behavioural or vegetative responses indicating that this patient fulfilled the typical criteria of pain insensitivity (9). The other somesthesic functions (tactile, thermoalqesic and deep) were normal. The osteotendinous reflexes were normal and symmetrical and no other anomaly was found on neurological examination. There was no sign of dysautonomy; no orthostatic hypotension, no anhydrosis or anomalies of the lachrymal or salivary secretions: pupils were symmetrical and reacted normally to light and pharmacological agents (neosynephrine, pilocarpine, tropicamide). The electrocardiogram showed a sinus rhythm and a normal baroreflex response to compression of the carotid and the Vasalva manoeuver. On the WAIS adult intelligence scale, verbal IQ was 92, performance IQ 96 and full-scale IQ 94. Personality examination did not show any sign of the psychiatric or epileptoid series (Roschach test).

Biological tests on blood and urine and examination of the CSF (cytochemistry, cytology, immunoqlobulin assay) did not reveal any anomaly. Skull X-ray showed only a slight internal frontal hyperostosis. Gas encephalography and CAT scanning indicated a moderate atrophy of the right sylvian area; however the size of the cerebral ventricles was normal.

Neurophysioloqical investigations

The effects of electrical stimulation on the perception threshold and on the withdrawal reflex were studied before and after the i.v. administration of naloxone (Winthrop; 0.8mq total dose dissolved in 125ml of isotonic glucose serum). Stimulation (15 set trains of rectangular pulses at 15Hz, supramaximal intensity, RACIA MEDELEC MS6) was performed via bipolar electrodes type MEDELEC E 212 M (distance between anode and cathode: 2.5mm) in contact with the soles of the feet (territory of the lateral planter nerve). The same test was carried out on the pulp of the left index finger (ring RACIA E DS). The axon reflex, neuro- muscular and cutaneous biopsy were not studied owing to refusal by the patient.

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Biochemical and pharmacological investigations Lumbar CSF was collected twice before naloxone treatment: at 3.00 PM on the sixth preceding day (sample 1) and at 10.00 AM on the day of treatment (sample 2), one min before starting the i.v. infusion. A third lumbar puncture (sample 3) was performed 8 min after the administration of the opiate antagonist. Samples were immediately acidified (with HCl up to O.lN final concentration), frozen on dry ice and stored at -20°C until assayed for MELM.

The CSF collected from 17 patients (8 males and 9 females suffering from multiple sclerosis) with no anomaly in pain and somesthetic perception were treated as above and used as controls. Since CSF endomorphin levels do not exhibit significant variations as a function of sex or age (lo), samples from control patients (n=5) were pooled for the chromatographic analysis (see below).

After thawing, samples were heated at 95*C for 1Omin and then centrifuged at 15,000 g for Smin. Aliquots (25-1OOul) of each supernatant (adjusted to pH 7.6 with 1M Tris base) were assayed in duplicate for their met-enkephalin (ME) content according to the radioimmunological method described in detail elsewhere (11). Among the numerous compounds tested (including leu-enkephalin, dynorphins and beta-endorphin, see ll), those cross-reacting in the assay were met-enkephalin-sulfoxide6(ME-SO, 360% as compared to 100% for ME) and met-enkephalin-Arg -Phe (ME-Arg-Phe, 19%). Biogel P2 chromatography was performed as described previously (11).

RESULTS

The present neurophysiological investigations confirmed the relative pain insensitivity since electrical stimulation of the sole using rectangular pulses of 0.5msec at 500 volts, which produced unendurable pain in normal subjects, was well withstood by the patient. Pain insensitivity was markedly reduced by naloxone since 10 min after the administration of the opiate antagonist, a less intense stimulation (rectangular pulses of 0.2msec at 250 volts) was enough to induce a sensation of pain.

When the electrical stimulation was applied to the finger pulp, only a withdrawal reflex with no pain could be induced using the following parameters: 15 set trains of rectangular pulses, 130 volts, supramaximal intensity, duration 0.2msec, frequency 1Hz. Ten min after naloxone injection, a nociceptive reflex with pain on flexion of the arm was induced by a less intense distal electrical stimulation (60 volts, 0.2msec, 1Hz).

In the patient's CSF (fig.lA,B) as well as in pooled control CSF (fig.lC), MELM could be separated by Biogel P2 chromatography into two fractions. The fraction I was excluded from the gel while the fraction II coeluted with ME-related peptides (ME-SO and ME-Arg-Phe) (fig.lA,B,C). Very little MELM was detected in the fractions corresponding to ME itself. The peak of immuno- reactivity which appeared between fractions I and II was in fact

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0 10 20 30 40 50 60 70

‘10 20 30 40 50 60 70

Fraction number

Figs 1A,B ,C

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FIG 1 Biogel P, chromatography of met-enkephalin-like

material ifi the CSF from the patient insensitive to pain (A,B). Comparison with CSF from control patients (C)

A: Aliquots (0.5ml) of CSF samples 1 and 2 taken before naloxone treatment were pooled and lyophilized. The dry residue was solubilized 10min;

with lml of 0.2N CH3COOH, centrifuged (4,000 g; 4“C) and the supernatant was filtered through a Biogel

P column (54cm high; t8e eluent.

0.9cm in diameter) with 0.2N CH3COOH as The collected fractions (lml) were lyophilized

and assayed for ME-like immunoreactivity (4). The void volume of the gel was elution volumes

measured wi&h cytochrome C (Cyt C). The of ME, ME-Arg -Phe and ME-SO were determined

with pure standards. The peak observed between fractions I and II corresponded to the elution of salts (--o--). Its apparent imrnunoreactivity resulted mainly (if not exclusively) from the non-specific interference of salts in the radioimmunoassay.

B: The CSF sample (lml) taken after naloxone treatment was treated and chromatographed as described in A.

c: The same protocol as that described in A was applied to lml of pooled CSF from control patients

artifactual since it corresponded to the elution of salts which interfered non specifically in the assay (see 11). The possible presence of ME-like substances in the salt eluates was not investigated.

Although the levels of total MELM in the three CSF samples collected from the patient were in the same range as those found in controls, quantitative differences were noted about the relative proportions of MELM in fractions I and II. Whereas 66% of MELM was eluted in fraction II of control CSF, only 46.5% of this material was found in the same fraction of the patient's CSF collected before naloxone treatment (table 1). This percentage was even lower (23.6%) after the administration of the opiate antagonist (table 1).

DISCUSSION

Radioreceptor, bio- and radio-immunoassays have been used to measure endomorphin levels in human CSF (1,2,4,12,13). Variations in the technical conditions of radioreceptor assays and in the specificity of the radioimmunoassays used by the different groups could explain why the reported values (expressed as ME equiva- lents) for "endorphin" or "met-enkephalin" concentrations in the CSF of control subjects varied between 13 (2) and 12,500 pg/ml (12). Unfortunately, such variations preclude any comparison of the absolute values found presently with those reported previously.

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Table 1

Met-enkephalin-like material

Total CSF Fraction I (%) Fraction II (%)

Patient insensitive to pain:

before naloxone' saqjle

'sample

.after naloxone sample

Control patients (n=17)

l_ 90.4 82.5 44.1 (53.5) 38.4 (46.5)

2 74.7

3 108.4 82.8 (76.4) 25.6 (23.6)

107.3217.9 36.5 (34.0) 70.8 (66.0)

Levels of met-enkephalin-like material in total CSF and fractions I and II from the patient insensitive to pain

compared to those from control patients Fractions I and II were separated using Biogel P, chromatography (see fig.1). Results are expressed in pg/ml of %E equivalents. The percentage of MELM found in each fraction is indicated in parentheses. The levels of MELM in total CSF from control patients are the mean 2 SEM of 17 independent determinations.

It has been mentioned recently that authentic ME is a minor component of receptor active CSF endorphins (13). The present chromatographic analyses confirmed this finding since ME did not contribute significantly to the radioimmunoassayable MELM detected in various CSF samples (fig.lA,B,C). Indeed, CSF MELM corresponded in part (about one third of total MELM in controls) to high molecular weight (MW >/ 2,000) compounds excluded from Biogel P2 (in fraction I). Previous investigations have demonstrated that these compounds are apparently neither ME- carriers nor ME-precursors (11). Furthermore, the high specifi- city of the ME-antiserum used for the radioimmunoassays permitted the exclusion of P-endorphin and dynorphins from those molecules accounting for MELM in fraction I. The remaining part of MELM (about two thirds of total in controls) was eluted fgom Bjogel P in the positions expected for ME-SO and ME-Arg -Phe . HPL?! analysis would be needed to identify the precise nature of this material. Its idpntity (at least partly) with ME-SO rather than with ME-Arg -Phe could be suspected on the basis of previous analyses of human (13) and cat (11) CSF.

As previously reported in similar cases of congenital insensiti- vity to pain (8), naloxone administration restored pain sensation

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and nociceptive reflex in our patient. Nevertheless, it cannot be automatically concluded that pain insensitivity resulted from the hyperactivity of an endomorphin system since normal levels of MELM were found in this particular patient. Several hypotheses can be put forward to explain this apparent paradox: 1. There was an increased release of MELM in the patient's CNS but CSF levels did not reflect the activated state for some unknown reasons (increased catabolism of MELM for instance). However previous investigations in cats (11) have shown that MELM levels rise markedly in the CSF under conditions producing an activation of central ME-containing neurones. 2. Enkephalinergic neurones were not involved in the present case of pain insensitivity and the naloxone effect resulted from the blockade of other activated endomorphin system(s). In this respect, it has to be emphasized that the relative proportion of (unidentified) MELM in fraction I was higher in the patient than in controls (table 1). The further increase noted after naloxone treatment (table 1) might be related to some feedback activating process triggered by the blockade of opiate receptors. Whether such modifications could also occur in normal subject after naloxone administration cannot be answered at present. Nevertheless, some hyperactivity of another (non-enkephalin) endomorphin system can still be suspected in the patient since she is epileptic and epilepsia can be triggered by opioid peptides (14). 3. Pain insensitivity was not the consequence of some anomaly in endomorphin systems but resulted from alterations in other neuronal population(s). Indeed, it has been shown recently that insensitivity to pain can be associated with a marked degenera- tion of substance P-containing axons in the substantia gelati- nosa (15). The blockade by naloxone of the inhibitory influence of endogenous opiates on afferent substance P fibres might facilitate the transfer of nociceptive messages (16). This would explain why naloxone treatment restored pain sensitivity in the patient.

In conclusion, this study shows that, in a case of congenital insensitivity to pain, the activity of ME-containing neurones is probably normal. Examination of other cases of this very uncommon disease will be necessary however before drawing any general conclusion about the possible lack of any functional relationship between MELM levels in the CSF and pain insensitivity.

ACKNOWLEDGEMENTS

This research was supported by grants from INSERM (PRC 120.039) and Rhane Poulenc. We are grateful to Dr M.D. Hall for critical reading of the manuscript.

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Received 2/2/84 Accepted 20/3/84

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