Neuroscience Letters, 77 (1987) 349 352 349

Elsevier Scientific Publishers Ireland Ltd.

NSL 04653

No gradient exists between lumbar and ventricular cerebrospinal fluid fl-endorphin

F. Facchinett i l, F. Petraglia 1, S. Cicero 2, G. N a p p i 3, M. Valentini I and A.R. G e n a z z a n i 1

1Department ~/" Ohstetrics and Gynecology, University o[" Modena, Modena (Italy), :Department ~[ Neurosurgery, University ~?f Catania, Catania (Italy) and 3Neurological lnstitute 'C. Mondino' University

~?/" Pavia, Pavia (Italy)

(Received 4 February 1987; Accepted 3 March 1987)

Key words." /J-Endorphin; ,#-Lipotropin; Adrenocorticotropic hormone; Gradient; Ventricular cerebro-

spinal fluid; Lumbar cerebrospinal fluid; Man

In 8 patients, fl-lipotropin (/]-LPH), fl-endorphin (fl-EP), ACTH, protein and chloride concentrations were measured in cerebrospinal fluid (CSF) samples obtained simultaneously from the lumbar space and lateral ventricle. Albumin and lgG levels were significantly higher in lumbar than in ventricular CSF sam-

ples while no craniocaudal gradient was observed for neuropeptide concentrations. The importance of

molecular weight in determining such a regional distribution is supported also by the similar lumbar and ventricular levels of chlorides. These data would validate the CSF approach through lumbar puncture as a tool for exploring neuropeptides in the central nervous system.

A growing number of studies discuss the role and significance of newly discovered neuropeptides. In humans, there are intrinsic limitations to efforts to understand the problem and the most common approach is evaluation of cerebrospinal fluid (CSF) levels of a given substance (the other being measurement of the tissue content of post- mortem brains).

There are, however, several methodological flaws in the CSF approach, mainly related to hydrodynamics, while the existence of a discrete distribution of proteins from ventricles to cisterna magna to the lumbar 'cul de sac' has been known for several years [8]. Since the lumbar CSF concentration of proopiomelanocortin- related peptides has been used by other researchers [I, 5, 6] as well as by ourselves [4, 7] as a reflection of central opioid content, we tested the possible existence of a gradient between spinal and ventricular CSF levels offl-endorphin (fl-EP),/~-lipotro- pin (fl-LPH) and ACTH.

The study was performed on 8 patients aged 2(~60 years, who had been admitted to the neurosurgical department of the University of Catania for increased intracra-

Corre,spondenee. F. Facchinetti, Clinica Ostetrica e Ginecologica dell'Universit~i di Modena, Via del Pozzo, 71, 1-41 I00 Modena, Italia.

0304-3940/87,,'$ 03.50 © 1987 Elsevier Scientific Publishers Ireland Ltd.

350

nial pressure due to hemorrhage or neoplasm. Four milliliters CSF were collected simultaneously from the second lumbar space and from the lateral ventricle at the moment of surgery. CSF was centrifuged, trasylol added (1000 kIU/ml) and 2-ml samples, immediately frozen, were freeze-dried. Samples were redissolved in 0.7 mi 0.1 M phosphate buffer, 0.1% bovine serum albumin (BSA) and 0.2 ml were used for ACTH RIA which was carried out using NIADDK reference standard and anti- serum (NIH, Bethesda, MD). Sensitivity was 1.0 fmol/tube. Intra-assay coefficient of variation was 9.2 _+ 1.9%. The residual 0.5 ml was applied on a Sephadex G-75 col- umn (1.5 x 45 cm) to separate fl-LPH from fl-EP, according to the relative retention of human purified fl-LPH (obtained from C.H. Li, S. Francisco, CA) and synthetic fl-EP (Organon, Oss, The Netherlands) which were 0.52 and 0.81 respectively. Two fractions of 16 ml were collected from each column, freeze-dried, redissolved in phos- phate buffer and submitted to a specific RIA. Anti-N-terminal fl-LPH serum (recog- nizing also },-LPH, but not fl-EP) and anti-C-terminal fl-EP serum (fully cross-react- ing with fl-LPH), used in the fl-LPH and fl-EP RIA respectively, were both supplied by Prof. C.H. Li.

Assay sensitivity was 1.0 fmol/tube for both RIAs. Intra-assay coefficients of varia- tions were 8.5 _+ 1.6 and 10.1 _+ 2.1% for fl-LPH and fl-EP respectively; interassay coef- ficients of variation were 12.9_+2.7 and 13.4-1-3.1%, Further details have been recently reported [4].

Albumin and IgG levels were determined by electrophoresis in 5 out of 8 cases, and in the same samples, chloride ion was also measured, using a titration assay.

Individual CSF neuropeptide concentrations are reported in Fig. 1. Although the mean values (±S.D. ; fmol/ml) are similar between lumbar and ventricular fl-EP

B-Endorphin B-Lipotropin ACTH

100 0

%

5o lo N

0 0 V L V L V L

Fig. I. Paired ventricular (V) and lumbar (g) CSF concentrations offl-EP, fl-LPH and ACTH.

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(89.7_+26.5 and 83.0+27.6, respectively), fl-LPH (38.2_+ 12.9 and 32.6+ 10.3) and

ACTH (11.0_+ 3.9 and 9.5 ± 2.8), the percent difference of paired neuropeptide con-

centrations measured at the two sites exceeded the intra-assay coefficients of varia- tion _+2 S.D. in almost every case.

There was no univocal trend, however, since lumbar vs ventricular fl-EP, fi-LPH and ACTH levels were higher in 5 subjects and lower in the remainder. Moreover, these differences appear to be distributed completely at random since in only two cases the 3 peptides were concomitantly higher in lumbar vs ventricular samples.

On the other hand, albumin (42.7_+26.7 mg/ml) and IgG (1 16.0_+ 11.2) levels mea- sured in lumbar samples were significantly higher than those measured at the ventri- cular level (7.5 _+ 6.3 and 4.8_+ 7.6 respectively, P < 0.01). The reproducibility of this finding in all subjects tested indicated a physiological-like CSF protein distribution.

The existence of such a lumbar/ventricular gradient for proteins but not for neuro-

peptides can probably be ascribed to the high molecular weight of the former and to a different circulating mechanism between craniocaudal regions of the central ner- vous system (simple diffusion of neuropeptides against active flow transport in the case of heavy proteins) [2]. The importance of molecular weight in determining a lum-

bar ventricular gradient is further sustained by the similar ventricular (120.8 ! 28.4) and lumbar (1 12.2_+ 12.9) concentrations of chloride. From another point of view,

lumbar CSF is far from the site of production and its composition could be modified

by exchanges with brain and blood. However, the observation that plasma fl-EP and /#LPH concentrations are much lower than in CSF and those of ACTH are similar

in the two compartments [7] makes an enrichment of neuropeptides in lumbar CSF unlikely. It should also be taken into account that only for substance P a probable spinal cord origin has been described [9].

Whatever the reasons may be, these data indicate that concentrations offl-EP and related peptides measured in lumbar CSF samples are similar to those found in the lateral ventricle, which would validate the approach through lumbar puncture as a

tool for exploring neuropeptides in humans. Of course, CSF concentrations of a

given neuropeptide are not necessarily related (either positively or negatively) to the parenchymal content.

This paper was partly supported by CNR, P.F. 'Medicina Preventiva e Riabilita- tiwt', SP "Controllo del Dolore'. We wish to thank Prof. C.H. Li (San Francisco, CA)

and Dr. S. Raiti of the N I A D D K (Bethesda, MD) for the supply of materials.

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logy, 26(1976) I S. 3 Facchinetti, F., Petraglia, F., Nappi, G., Martignoni, E., Antoni, G., Parrini, D. and Genazzani, A.R.,

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4 Facchinetti, F,, Nappi, G., Petraglia, F., Martignoni, E., Sinforiani, E. and Genazzani, A.R., Central ACTH deficit in degenerative and vascular dementia, Life Sci., 35 (1984) 1691 1697.

5 Gerner, R.H. and Sharp, B., CSF fl-endorphin-immunoreactivity in normal, schizophrenic, depressed, manic and anorexic subjects, Brain Res., 237 (1982) 244-247.

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6 Jeffcoate, W.J., Rees, L.H.,. McLoughlin, L., Ratter, S.J., Hope, P.J., Lowry, J. and Besser, G.M.,/¢-EP in human cerebrospinal fluid, Lancet, i (1978) 119 121.

7 Nappi, G., Petraglia, F., Martignoni, E., Facchinetti, F., Bono, G. and Genazzani, A.R., fl-Endorphin cerebrospinal fluid decrease in untreated parkinsonian patients, Neurology, 35 (1985) 1371 1374.

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9 Wood, J.H., Neuronendocrinology of cerebrospinal fluid, peptides, steroids and other hormones, Neurosurgery, 11 (1982) 293305.