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beta-lipotropin immunoreactivity in human CSF
Life Sciences, Vol. 43, pp. 1545-1550 Printed in the U.S.A.
Pergamon Press
A G E - R E L A T E D C H A N G E S OF M E T H I O N I N E - E N K E P H A L I N AND B E T A - E N D O R P H I N BETA-LIPOTROPIN I M M U N O R E A C T I V I T Y IN H U M A N CSF Alessio,
L.,
Govoni,
S.*, Salar, G., Battaini, and Trabucchi, M . * *
F.**,Iavicoli,
/
R. A
D e p a r t m e n t s of N e u r o t r a u m a t o l o g y , and A N e u r o r a d i o l o g y , University of Padua, *Dept. of P h a r m a c o b i o l o g y U n i v e r s i t y of Bari, **Chair of Toxicology, 2nd U n i v e r s i t y of Rome, Italy. (Received in final form September 12, 1988) Summary Methionine-enkephalin (ME-IR) and b e t a - e n d o r p h i n (BE-IR) immunoreactive material CSF concentrations have been m e a s u r e d in s u b j e c t s of d i f f e r e n t ages a f f e c t e d by lumbar or cervical disk hernia. The two peptides exhibited different age-related trends. ME-IR levels rose significantly w i t h age while no changes were observed in the case of BE-IR.
In studies on the role of e n d o g e n o u s opioid peptides in pathophysiological conditions, the normal r e l a t i o n s h i p between levels of these p e p t i d e s and age is an i m p o r t a n t consideration. Changes in opioid peptides levels during aging have been demonstrated in v a r i o u s b r a i n areas: in 25 m o n t h aged rats, a decreased content of M E - I R was found in the striatum, in the cortex and in the spinal cord, but not in the hypothalamus (1,2,3). B E - I R c o n t e n t was also found decreased, in p a r t i c u l a r in the h y p o t h a l a m u s and in the p e r i a c q u e d u c t a l gray. In contrast, the c o n t e n t of b o t h M E - I R and B E - I R was g r e a t e r in the p i t u i t a r y of 25 m o n t h o l d rats r e s p e c t to c o n t r o l s (4). Post mortem s t u d i e s on h u m a n t i s s u e s h a v e shown either no changes or a r e d u c e d p e p t i d e c o n t e n t (5). AS h u m a n t i s s u e s are not e a s i l y available, the d e t e c t i o n of o p l o i d p e p t l d e c o n t e n t in biological fluids offers the p o s s i b i l i t y to evaluate opiate changes d u r i n g aging in man. A l o n g this line the p r e s e n t study a n a l y s e s the c o n t e n t of B E - I R and M E - I R in the CSF of p a t i e n t s of d i f f e r e n t ages.
Methods A gated.
s e r i e s of 61 p a t i e n t s (35 males, In all of t h e m M E - I R CSF levels
26 females) was investiwere determined. In a
0024-$205/88 $3.00 + .00 Copyright (c) 1988 Pergamon P r e s s p l e
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Aging and CSF ME and BE
Vol. 43, No. 19, 1988
subgroup of 17 p a t i e n t s also B E - I R c o n c e n t r a t i o n s w e r e measured. S a m p l e s w e r e c o l l e c t e d in the D e p a r t m e n t of N e u r o r a d i o l o g y of the P a d u a C i t y H o s p i t a l , from p a t i e n t s a d m i t t e d w i t h the d i a g n o s i s of l u m b a r or c e r v i c a l d i s k hernia; 60% of t h e m w e r e s u f f e r i n g from a chronic pain syndrome w h i l e the o t h e r s d i d not present pain symptomatology. In b o t h g r o u p s a s i g n i f i c a n t p r o p o r t i o n of the p a t i e n t s (80%) p r e s e n t e d p a r e s t h e s i a s . The age r a n g e d from 23 to 68 years. Patient's pharmaco therapy w a s l i m i t e d to analgesic aspirin-like a g e n t s and to a n t i a c i d and a n t i s e c r e t o r y drugs in ulcerous subjects. F o u r to e i g h t ml of c e r e b r o s p i n a l fluid were t a k e n f r o m e a c h p a t i e n t b e t w e e n 8.00 a.m. to 1.30 p.m. through lumbar puncture. The CSF was withdrawn in the course of radiographic d i a g n o s t i c p r o c e d u r e s r e q u e s t e d b y the n e u r o s u r g e o n or the n e u r o l o g i s t . I m m e d i a t e l y after w i t h d r a w a l samples were f r o z e n and k e p t at - 8 0 ° C until the time of assay. E x t r a c t i o n and R a d i o i m m u n o a s s a y o f M e t h i o n i n e - E n k e p h a l i n In order to remove i n t e r f e r e n c e in the RIA by proteins p r e s e n t in the C S F and to m i n i m i z e proteolysis, enkephalin-like p e p t i d e s w e r e e x t r a c t e d u s i n g a c e t o n e a c i d i f i e d w i t h HCl (0.01 N) f o l l o w i n g the p r o c e d u r e d e s c r i b e d by H e x u m et al. (6) and Govoni et al. (7) for plasma. In brief, four v o l u m e s of c o l d acetone were added to one v o l u m e of CSF ( r o u t i n e l y 2 ml) and placed o v e r n i g h t at 4 ~ . A f t e r this period, the m i x t u r e w a s c e n t r i f u g e d at 2 , 0 0 0 x g for i0 m i n u t e s . The s u p e r n a t a n t was p o u r e d off and the pellet re-extracted with a volume of acidified acetoned i s t i l l e d w a t e r (80:20, V:V). A f t e r 1 h o u r at 4°C, the samples were c e n t r i f u g e d at 5,000 x g for i0 minutes. The supernatants were combined a n d the a c e t o n e e x t r a c t e d w i t h petroleum ether. Residual a c e t o n e w a s r e m o v e d u n d e r r e d u c e d p r e s s u r e in a v a c u u m concentrator (Speed Vac). The extract was lyophilized, r e d i s s o l v e d in water, h e a t e d at 100°C, c e n t r i f u g e d at 12,000 x g for i0 minutes and assayed for peptide content. For each e x t r a c t i o n r e c o v e r y w a s e v a l u a t e d b y a d d i n g to a pool of control CSF a known amount of synthetic methionine-enkephalin. The r e c o v e r y r a n g e d f r o m 67 to 78% of the a d d e d peptide. Antibody was commercially supplied by UCB Bioproducts S.A. (Peptide Dept. Brussels, Belgium). The antiserum shows the following cross reactivities: 100% w i t h T y r - G l y - G l y - P h e - M e t , 7% w i t h T y r - G l y - G l y - P h e - L e u and 0 . 1 % w i t h G l y - P h e - M e t , Giy-Gly-PheMet, Tyr-Gly-Gly-Phe-Met-Arg-Phe, a l f a and b e t a - e n d o r p h i n , ACTH, beta-lipotropin. [ 125I]-methionine-enkephalin was prepared according to M i l l e r et al. (8). The s y n t h e t i c peptide, or the extract, was incubated at 4 °C o v e r n i g h t w i t h a n t i s e r u m and [125I] m e t h i o n i n e - e n k e p h a l i n in 1 ml of 0.2 M t r i s b u f f e r pH 7.4 containing 0.1% bovine serum albumin and 0.15% dextran. The antibody b o u n d 125 I - p e p t i d e w a s s e p a r a t e d f r o m the free peptide by a d d i t i o n of 0.2 ml of 1.5% c h a r c o a l s l u r r y containing 0.15% d e x t r a n and 0 . 9 % NaCI. A f t e r c e n t r i f u g a t i o n , the s u p e r n a t a n t was decanted in a s c i n t i l l a t i o n vial and the s a m p l e c o u n t e d in a Gamma-counter. Assay detection limit w a s 5 pg of synthetic methionine-enkephalin. Usually samples were run in triplicate which varied by less than 5%. Interassay coefficient of
Vol. 43, No. 19, 1988
variation
was
Aging and CSF ME and BE
1547
10.2%.
Radioimmunoassayof Beta-Endorphin For r o u t i n e a s s a y l i q u o r s a m p l e s ~one ml.) w e r e t r e a t e d for i0 min. at i00 C and t h e n lyophilized. The samples were reconstituted in 0.3 ml of buffer and aliquots of 0.i ml radioimmunoassayed. Beta-endorphin immunoreactivity was measured using c o m m e r c i a l l y s u p p l i e d R I A k i t s p r o v i d e d by N E N (USA). The antiserum presents the f o l l o w i n g c r o s s reactivities on molar basis: b e t a - e n d o r p h i n 100%, b e t a - L P H 50%, a l p h a - e n d o r p h i n 0.01%, l e u c i n e - e n k e p h a l i n 0.004%, m e t h i o n i n e - e n k e p h a l i n 0.004%, b e t a - M S H 0.01%. The i n t r a - and inter-assay variation coefficients were less than 5% and 10% respectively. Due to antiserum crossreactivity w i t h b e t a - L i p o t r o p i n the i m m u n o r e a c t i v e m a t e r i a l w i l l be r e f e r r e d as B E / B L P H - I R H P L C of M e t h i o n i n e - E n k e p h a l i n Following lyophilization CSF extracts were reconstituted in i00 ul of 1 M p y r i d i n e / 0.5 M a c e t i c a c i d and a p p l i e d to a HPLC reverse p h a s e B i o - S i l ODS I0 c o l u m n (250 x 4 mm). The material was e l u t e d f r o m the c o l u m n w i t h a g r a d i e n t of l - p r o p a n o l in 1 M pyridine/0.5 M acetic a c i d (0 to 50 %) at 24 ml/hr. One ml fractions were collected, lyophilized, r e s u s p e n d e d in d i s t i l l e d water and radioimmunoassayed for methionine-enkephalin as d e s c r i b e d (7). Results
and D i s c u s s i o n
T a b l e 1 r e p o r t s the C S F M E - I R c o n c e n t r a t i o n s in the patients d i v i d e d a c c o r d i n g to t h e i r age in two s u b g r o u p s : A: < 60 years; B: ~ 60 years. The m e a n M E - I R CSF v a l u e w a s h i g h e r in g r o u p B in c o m p a r i s o n w i t h g r o u p A.
TABLE
i
Methionine-Enkephalin and B e t a - E n d o r p h i n I m m u n o r e a c t i v e in C S F as a F u n c t i o n of A g e
GROUP ( m e a n age)
N
AGE
ME-IR (pg/ml)
N .
A: < 60 y e a r s (44.0Z8.4)
44
67.8+8.1
B:
17
80.5+6.6*
> 60 y e a r s
(62.8~8.1) Values
are
.
.
.
.
BE/BLPH-IR (pg/ml) .
.
.
12
5
-means
* P < 0.001, Moreover
+
S.D.
Student a highly
t
:
5.77
Material
.
.
.
.
.
.
.
.
.
.
.
.
.
62.4+16.1
59.9+
6.9
-df
59.
t-test. significant
linear
correlation
between ME-IR
1548
Aging and CSF ME and BE
a n d age w a s o b s e r v e d
Vol. 43, No. 19, 1988
( F i g u r e i).
IOO 90.
a
Z
m m
v
70 I
Z
°
°~ o ~
60.
50
48
20
30 Age
(yea~)
Figure ! C o r r e l a t i o n B e t w e e n A g e a n d M E - I R C o n t e n t in H u m a n CSF. Correlation plot was generated b y the m e t h o d of the least squares analysis; Y = 0 . 5 5 5 X + 43.8; N = 61, R = 0.659, P < 0 . 0 0 1
A pool of C S F w a s e x t r a c t e d , l y o p h i l i z e d and s u b j e c t e d to HPLC reverse phase chromatography. U s i n g the a d o p t e d extraction procedure the i m m u n o r e a c t i v e m a t e r i a l d e t e c t e d in the CSF e l u t e s as a s i n g l e p e a k of i m m u n o r e a c t i v i t y in the same p o s i t i o n as a s y n t h e t i c m e t h i o n i n e - e n k e p h a l i n s t a n d a r d ( r e t e n t i o n t i m e 45 min). The i m m u n o r e a c t i v e m a t e r i a l d e t e c t e d in CSF r e p r e s e n t s t h e r e f o r e m e t h i o n i n e - e ~ k e p h a l i n , a l t h o u g h the a n a l y s i s does n o t e x c l u d e the p r e s e n c e of o t h e r e n k e p h a l i n - l i k e s e q u e n c e s not r e c o g n i z e d by our antibody. B E / B L P H - I R c o n c e n t r a t i o n s d i d not d i s p l a y significant age-related changes at least in the range of ages i n v e s t i g a t e d . ( T a b , i). Patients e x a m i n e d w e r e a f f e c t e d by c e r v i c a l a n d l u m b a r disk hernia; no significant differences were found between patients w i t h or w i t h o u t pain. No r e l a t i o n w a s f o u n d b e t w e e n the l e n g h t of p a i n s y m p t o m a t o l o g y ( r a n g i n g f r o m 15 d a y s to 5 y e a r s ) and p e p t i d e levels, both in the c a s e of M E - I R and of BE-IR. No c o r r e l a t i o n was found by multioriented analysis between peptides levels and o t h e r p a r a m e t e r s s u c h as sex, b l o o d p r e s s u r e , b o d y w e i g h t . In p r e v i o u s s t u d i e s p e r f o r m e d on p l a s m a (9,10) no c o r r e l a t i o n between M E - I R and B E - I R l e v e l s and age w a s found; o n the other hand, to our k n o w l e d g e no d a t a are r e p o r t e d in literature on
Vol. 43, No. 19, 1988
Aging and CSF ME and BE
1549
opiate C S F c h a n g e s d u r i n g a g i n g e x c e p t for a s t u d y m a d e b y Bach et al. (ii) in w h i c h a d e c r e a s e of B E - I R CSF c o n t e n t w i t h age was demonstrated. D i f f e r e n c e s in m e t h o d o l o g y , in p a r t i c u l a r the lack of s e p a r a t i o n in o u r a s s a y c o n d i t i o n s of BE and BLPH, as well as in the r a n g e of ages i n v e s t i g a t e d (in the B E - I R g r o u p the older p a t i e n t in the p r e s e n t s t u d y is 66 y e a r old) m a y a c c o u n t for this discrepancy. The l i m i t e d n u m b e r of C S F s a m p l e s in w h i c h B E / B L P H IR w a s a s s a y e d m a y also e x p l a i n the lack of a g e - r e l a t e d changes in C S F B E / B L P H - I R c o n t e n t in the p r e s e n t study. Animal data s h o w i n g no i n c r e a s e of the methionine-enkephalin encephalic c o n t e n t w i t h age s e e m to e x c l u d e the p o s s i b i l i t y that the CSF c h a n g e s are due to an increased methionine-enkephalin s y n t h e s i s at e n c e p h a l i c levels. On the o t h e r h a n d a d e c r e a s e d a c t i v i t y of p e p t i d e degrading e n z y m e s in C S F c o u l d be the c a u s e of the r a i s e d M E - I R CSF levels in aged people; a l t h o u g h the o b s e r v a t i o n that beta-endorphin levels are u n m o d i f i e d (this study) or d e c r e a s e d ( ii ) s e e m s to exclude this hypothesis. The a s s a y of b o t h u n s p e c i f i c peptide degrading e n z y m e s a c t i v i t y as well as of s p e c i f i c enkephalinase activity in l i q u o r or spinal c o r d t i s s u e w o u l d be helpful to clarify this point. In a d d i t i o n the s t a b i l i t y of methioninee n k e p h a l i n a n d b e t a - e n d o r p h i n in CSF c a n be c o n v e n i e n t l y studied u s i n g r a d i o l a b e l l e d m e t h i o n i n e - e n k e p h a l i n and b e t a - e n d o r p h i n . W h e t h e r the i n c r e a s e d CSF M E - I R l e v e l s are due to an e n h a n c e d synthesis a n d r e l e a s e at spinal s i t e s or to a l o w e r t u r n o v e r of the p e p t i d e in C S F of o l d e r p a t i e n t s has to be a s c e r t a i n e d . The i n c r e a s e in C S F M E - I R m a y be r e l a t e d to the o b s e r v e d age-related changes in p a i n t h r e s h o l d and n a r c o t i c a n a l g e s i c effect which h a v e b e e n n o t i c e d in g e r i a t r i c p a t i e n t s (12,13).
References i.C. MISSALE, S. GOVONI, L. C A S T E L L E T T I , P . F . S P A N O and M. T R A B U C C H I , in: A . A g n o l i et Ai.Eds., A g i n g B r a i n and Ergot A l k a l o i d s , A g i n g Voi.23, R a v e n P r e s s pp: 15-22, (1983). 2. C. M I S S A L E , S. GOVONI, L. CASTELLETTI, P.F. S P A N O AND M. T R A B U C C H I , B r a i n Res. 262, 160-162, (1983). 3. A. DUPONT, P. SANARD, Y. MACAND, F. L A B R I E and J.K. BOISSIER, L i f e S c i e n c e s , 29, 2317-2322, (1981). 4. C. MISSALE, S. GOVONI, L. CROCE, A. BOSIO, P.F. S P A N O and M. T R A B U C C H I , J. N e u r o c h e m . , 40, 20-24, (1983). 5. O. HORNYKIEWICZ, Modification of cell to cell signals d u r i n g n o r m a l and p a t h o l o g i c a l aging. ( Eds S. G O V O N I and F. B A T T A I N I ) S p r i n g e r V e r l a g pp. 169-182, (1987). 6. T.D. H E X U M , I. H A N B A U E R , S. GOVONI, H.Y.T. YANG, and E.COSTA, N e u r o p e p t i d e s , l, 137-142, (1980). 7. S . G O V O N I , I. H A N B A U E R , T.D. HEXUM, H.Y.T. YANG, G.D. K E L L Y and E.COSTA, Neuropharmacology, 20, 639-645, (1981). 8. R . J . M I L L E R , R.H.CHANG, I. LEIGHTAN and P . Q U A T R E C A S A S , Life Sci., 22, 379-388, (1978). 9. S. GOVONI, G. PASINETTI, A. BIANCHI, M. GADOLA and M. T R A B U C C H I , A l c o h o l and D r u g Res., Z, 93-98, (1986).
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i0. S. GOVONI, G. PASINETTI, M.R. INZOLI, R. ROZZINI and M. TRABUCCHI, Life Sci., 35, 2594-2552, (1984). ii. F.W. BACH, K.JENSEN, N.BLEGUAD, M.FENGER, R . J O R D A L and J. OLESEN, Cephalalgia, 5, 23-27, (1983). 12. J . W . B E L V I L L E , W.H. FORREST, E. M I L L E R and B.W.BROWN, JAMA, 217, 1835-1841, (1971). 13. R.F.KAIKO, Clin. Pharmacol. Ther., 2_88, 832-826. (1980).
Pergamon Press
A G E - R E L A T E D C H A N G E S OF M E T H I O N I N E - E N K E P H A L I N AND B E T A - E N D O R P H I N BETA-LIPOTROPIN I M M U N O R E A C T I V I T Y IN H U M A N CSF Alessio,
L.,
Govoni,
S.*, Salar, G., Battaini, and Trabucchi, M . * *
F.**,Iavicoli,
/
R. A
D e p a r t m e n t s of N e u r o t r a u m a t o l o g y , and A N e u r o r a d i o l o g y , University of Padua, *Dept. of P h a r m a c o b i o l o g y U n i v e r s i t y of Bari, **Chair of Toxicology, 2nd U n i v e r s i t y of Rome, Italy. (Received in final form September 12, 1988) Summary Methionine-enkephalin (ME-IR) and b e t a - e n d o r p h i n (BE-IR) immunoreactive material CSF concentrations have been m e a s u r e d in s u b j e c t s of d i f f e r e n t ages a f f e c t e d by lumbar or cervical disk hernia. The two peptides exhibited different age-related trends. ME-IR levels rose significantly w i t h age while no changes were observed in the case of BE-IR.
In studies on the role of e n d o g e n o u s opioid peptides in pathophysiological conditions, the normal r e l a t i o n s h i p between levels of these p e p t i d e s and age is an i m p o r t a n t consideration. Changes in opioid peptides levels during aging have been demonstrated in v a r i o u s b r a i n areas: in 25 m o n t h aged rats, a decreased content of M E - I R was found in the striatum, in the cortex and in the spinal cord, but not in the hypothalamus (1,2,3). B E - I R c o n t e n t was also found decreased, in p a r t i c u l a r in the h y p o t h a l a m u s and in the p e r i a c q u e d u c t a l gray. In contrast, the c o n t e n t of b o t h M E - I R and B E - I R was g r e a t e r in the p i t u i t a r y of 25 m o n t h o l d rats r e s p e c t to c o n t r o l s (4). Post mortem s t u d i e s on h u m a n t i s s u e s h a v e shown either no changes or a r e d u c e d p e p t i d e c o n t e n t (5). AS h u m a n t i s s u e s are not e a s i l y available, the d e t e c t i o n of o p l o i d p e p t l d e c o n t e n t in biological fluids offers the p o s s i b i l i t y to evaluate opiate changes d u r i n g aging in man. A l o n g this line the p r e s e n t study a n a l y s e s the c o n t e n t of B E - I R and M E - I R in the CSF of p a t i e n t s of d i f f e r e n t ages.
Methods A gated.
s e r i e s of 61 p a t i e n t s (35 males, In all of t h e m M E - I R CSF levels
26 females) was investiwere determined. In a
0024-$205/88 $3.00 + .00 Copyright (c) 1988 Pergamon P r e s s p l e
1546
Aging and CSF ME and BE
Vol. 43, No. 19, 1988
subgroup of 17 p a t i e n t s also B E - I R c o n c e n t r a t i o n s w e r e measured. S a m p l e s w e r e c o l l e c t e d in the D e p a r t m e n t of N e u r o r a d i o l o g y of the P a d u a C i t y H o s p i t a l , from p a t i e n t s a d m i t t e d w i t h the d i a g n o s i s of l u m b a r or c e r v i c a l d i s k hernia; 60% of t h e m w e r e s u f f e r i n g from a chronic pain syndrome w h i l e the o t h e r s d i d not present pain symptomatology. In b o t h g r o u p s a s i g n i f i c a n t p r o p o r t i o n of the p a t i e n t s (80%) p r e s e n t e d p a r e s t h e s i a s . The age r a n g e d from 23 to 68 years. Patient's pharmaco therapy w a s l i m i t e d to analgesic aspirin-like a g e n t s and to a n t i a c i d and a n t i s e c r e t o r y drugs in ulcerous subjects. F o u r to e i g h t ml of c e r e b r o s p i n a l fluid were t a k e n f r o m e a c h p a t i e n t b e t w e e n 8.00 a.m. to 1.30 p.m. through lumbar puncture. The CSF was withdrawn in the course of radiographic d i a g n o s t i c p r o c e d u r e s r e q u e s t e d b y the n e u r o s u r g e o n or the n e u r o l o g i s t . I m m e d i a t e l y after w i t h d r a w a l samples were f r o z e n and k e p t at - 8 0 ° C until the time of assay. E x t r a c t i o n and R a d i o i m m u n o a s s a y o f M e t h i o n i n e - E n k e p h a l i n In order to remove i n t e r f e r e n c e in the RIA by proteins p r e s e n t in the C S F and to m i n i m i z e proteolysis, enkephalin-like p e p t i d e s w e r e e x t r a c t e d u s i n g a c e t o n e a c i d i f i e d w i t h HCl (0.01 N) f o l l o w i n g the p r o c e d u r e d e s c r i b e d by H e x u m et al. (6) and Govoni et al. (7) for plasma. In brief, four v o l u m e s of c o l d acetone were added to one v o l u m e of CSF ( r o u t i n e l y 2 ml) and placed o v e r n i g h t at 4 ~ . A f t e r this period, the m i x t u r e w a s c e n t r i f u g e d at 2 , 0 0 0 x g for i0 m i n u t e s . The s u p e r n a t a n t was p o u r e d off and the pellet re-extracted with a volume of acidified acetoned i s t i l l e d w a t e r (80:20, V:V). A f t e r 1 h o u r at 4°C, the samples were c e n t r i f u g e d at 5,000 x g for i0 minutes. The supernatants were combined a n d the a c e t o n e e x t r a c t e d w i t h petroleum ether. Residual a c e t o n e w a s r e m o v e d u n d e r r e d u c e d p r e s s u r e in a v a c u u m concentrator (Speed Vac). The extract was lyophilized, r e d i s s o l v e d in water, h e a t e d at 100°C, c e n t r i f u g e d at 12,000 x g for i0 minutes and assayed for peptide content. For each e x t r a c t i o n r e c o v e r y w a s e v a l u a t e d b y a d d i n g to a pool of control CSF a known amount of synthetic methionine-enkephalin. The r e c o v e r y r a n g e d f r o m 67 to 78% of the a d d e d peptide. Antibody was commercially supplied by UCB Bioproducts S.A. (Peptide Dept. Brussels, Belgium). The antiserum shows the following cross reactivities: 100% w i t h T y r - G l y - G l y - P h e - M e t , 7% w i t h T y r - G l y - G l y - P h e - L e u and 0 . 1 % w i t h G l y - P h e - M e t , Giy-Gly-PheMet, Tyr-Gly-Gly-Phe-Met-Arg-Phe, a l f a and b e t a - e n d o r p h i n , ACTH, beta-lipotropin. [ 125I]-methionine-enkephalin was prepared according to M i l l e r et al. (8). The s y n t h e t i c peptide, or the extract, was incubated at 4 °C o v e r n i g h t w i t h a n t i s e r u m and [125I] m e t h i o n i n e - e n k e p h a l i n in 1 ml of 0.2 M t r i s b u f f e r pH 7.4 containing 0.1% bovine serum albumin and 0.15% dextran. The antibody b o u n d 125 I - p e p t i d e w a s s e p a r a t e d f r o m the free peptide by a d d i t i o n of 0.2 ml of 1.5% c h a r c o a l s l u r r y containing 0.15% d e x t r a n and 0 . 9 % NaCI. A f t e r c e n t r i f u g a t i o n , the s u p e r n a t a n t was decanted in a s c i n t i l l a t i o n vial and the s a m p l e c o u n t e d in a Gamma-counter. Assay detection limit w a s 5 pg of synthetic methionine-enkephalin. Usually samples were run in triplicate which varied by less than 5%. Interassay coefficient of
Vol. 43, No. 19, 1988
variation
was
Aging and CSF ME and BE
1547
10.2%.
Radioimmunoassayof Beta-Endorphin For r o u t i n e a s s a y l i q u o r s a m p l e s ~one ml.) w e r e t r e a t e d for i0 min. at i00 C and t h e n lyophilized. The samples were reconstituted in 0.3 ml of buffer and aliquots of 0.i ml radioimmunoassayed. Beta-endorphin immunoreactivity was measured using c o m m e r c i a l l y s u p p l i e d R I A k i t s p r o v i d e d by N E N (USA). The antiserum presents the f o l l o w i n g c r o s s reactivities on molar basis: b e t a - e n d o r p h i n 100%, b e t a - L P H 50%, a l p h a - e n d o r p h i n 0.01%, l e u c i n e - e n k e p h a l i n 0.004%, m e t h i o n i n e - e n k e p h a l i n 0.004%, b e t a - M S H 0.01%. The i n t r a - and inter-assay variation coefficients were less than 5% and 10% respectively. Due to antiserum crossreactivity w i t h b e t a - L i p o t r o p i n the i m m u n o r e a c t i v e m a t e r i a l w i l l be r e f e r r e d as B E / B L P H - I R H P L C of M e t h i o n i n e - E n k e p h a l i n Following lyophilization CSF extracts were reconstituted in i00 ul of 1 M p y r i d i n e / 0.5 M a c e t i c a c i d and a p p l i e d to a HPLC reverse p h a s e B i o - S i l ODS I0 c o l u m n (250 x 4 mm). The material was e l u t e d f r o m the c o l u m n w i t h a g r a d i e n t of l - p r o p a n o l in 1 M pyridine/0.5 M acetic a c i d (0 to 50 %) at 24 ml/hr. One ml fractions were collected, lyophilized, r e s u s p e n d e d in d i s t i l l e d water and radioimmunoassayed for methionine-enkephalin as d e s c r i b e d (7). Results
and D i s c u s s i o n
T a b l e 1 r e p o r t s the C S F M E - I R c o n c e n t r a t i o n s in the patients d i v i d e d a c c o r d i n g to t h e i r age in two s u b g r o u p s : A: < 60 years; B: ~ 60 years. The m e a n M E - I R CSF v a l u e w a s h i g h e r in g r o u p B in c o m p a r i s o n w i t h g r o u p A.
TABLE
i
Methionine-Enkephalin and B e t a - E n d o r p h i n I m m u n o r e a c t i v e in C S F as a F u n c t i o n of A g e
GROUP ( m e a n age)
N
AGE
ME-IR (pg/ml)
N .
A: < 60 y e a r s (44.0Z8.4)
44
67.8+8.1
B:
17
80.5+6.6*
> 60 y e a r s
(62.8~8.1) Values
are
.
.
.
.
BE/BLPH-IR (pg/ml) .
.
.
12
5
-means
* P < 0.001, Moreover
+
S.D.
Student a highly
t
:
5.77
Material
.
.
.
.
.
.
.
.
.
.
.
.
.
62.4+16.1
59.9+
6.9
-df
59.
t-test. significant
linear
correlation
between ME-IR
1548
Aging and CSF ME and BE
a n d age w a s o b s e r v e d
Vol. 43, No. 19, 1988
( F i g u r e i).
IOO 90.
a
Z
m m
v
70 I
Z
°
°~ o ~
60.
50
48
20
30 Age
(yea~)
Figure ! C o r r e l a t i o n B e t w e e n A g e a n d M E - I R C o n t e n t in H u m a n CSF. Correlation plot was generated b y the m e t h o d of the least squares analysis; Y = 0 . 5 5 5 X + 43.8; N = 61, R = 0.659, P < 0 . 0 0 1
A pool of C S F w a s e x t r a c t e d , l y o p h i l i z e d and s u b j e c t e d to HPLC reverse phase chromatography. U s i n g the a d o p t e d extraction procedure the i m m u n o r e a c t i v e m a t e r i a l d e t e c t e d in the CSF e l u t e s as a s i n g l e p e a k of i m m u n o r e a c t i v i t y in the same p o s i t i o n as a s y n t h e t i c m e t h i o n i n e - e n k e p h a l i n s t a n d a r d ( r e t e n t i o n t i m e 45 min). The i m m u n o r e a c t i v e m a t e r i a l d e t e c t e d in CSF r e p r e s e n t s t h e r e f o r e m e t h i o n i n e - e ~ k e p h a l i n , a l t h o u g h the a n a l y s i s does n o t e x c l u d e the p r e s e n c e of o t h e r e n k e p h a l i n - l i k e s e q u e n c e s not r e c o g n i z e d by our antibody. B E / B L P H - I R c o n c e n t r a t i o n s d i d not d i s p l a y significant age-related changes at least in the range of ages i n v e s t i g a t e d . ( T a b , i). Patients e x a m i n e d w e r e a f f e c t e d by c e r v i c a l a n d l u m b a r disk hernia; no significant differences were found between patients w i t h or w i t h o u t pain. No r e l a t i o n w a s f o u n d b e t w e e n the l e n g h t of p a i n s y m p t o m a t o l o g y ( r a n g i n g f r o m 15 d a y s to 5 y e a r s ) and p e p t i d e levels, both in the c a s e of M E - I R and of BE-IR. No c o r r e l a t i o n was found by multioriented analysis between peptides levels and o t h e r p a r a m e t e r s s u c h as sex, b l o o d p r e s s u r e , b o d y w e i g h t . In p r e v i o u s s t u d i e s p e r f o r m e d on p l a s m a (9,10) no c o r r e l a t i o n between M E - I R and B E - I R l e v e l s and age w a s found; o n the other hand, to our k n o w l e d g e no d a t a are r e p o r t e d in literature on
Vol. 43, No. 19, 1988
Aging and CSF ME and BE
1549
opiate C S F c h a n g e s d u r i n g a g i n g e x c e p t for a s t u d y m a d e b y Bach et al. (ii) in w h i c h a d e c r e a s e of B E - I R CSF c o n t e n t w i t h age was demonstrated. D i f f e r e n c e s in m e t h o d o l o g y , in p a r t i c u l a r the lack of s e p a r a t i o n in o u r a s s a y c o n d i t i o n s of BE and BLPH, as well as in the r a n g e of ages i n v e s t i g a t e d (in the B E - I R g r o u p the older p a t i e n t in the p r e s e n t s t u d y is 66 y e a r old) m a y a c c o u n t for this discrepancy. The l i m i t e d n u m b e r of C S F s a m p l e s in w h i c h B E / B L P H IR w a s a s s a y e d m a y also e x p l a i n the lack of a g e - r e l a t e d changes in C S F B E / B L P H - I R c o n t e n t in the p r e s e n t study. Animal data s h o w i n g no i n c r e a s e of the methionine-enkephalin encephalic c o n t e n t w i t h age s e e m to e x c l u d e the p o s s i b i l i t y that the CSF c h a n g e s are due to an increased methionine-enkephalin s y n t h e s i s at e n c e p h a l i c levels. On the o t h e r h a n d a d e c r e a s e d a c t i v i t y of p e p t i d e degrading e n z y m e s in C S F c o u l d be the c a u s e of the r a i s e d M E - I R CSF levels in aged people; a l t h o u g h the o b s e r v a t i o n that beta-endorphin levels are u n m o d i f i e d (this study) or d e c r e a s e d ( ii ) s e e m s to exclude this hypothesis. The a s s a y of b o t h u n s p e c i f i c peptide degrading e n z y m e s a c t i v i t y as well as of s p e c i f i c enkephalinase activity in l i q u o r or spinal c o r d t i s s u e w o u l d be helpful to clarify this point. In a d d i t i o n the s t a b i l i t y of methioninee n k e p h a l i n a n d b e t a - e n d o r p h i n in CSF c a n be c o n v e n i e n t l y studied u s i n g r a d i o l a b e l l e d m e t h i o n i n e - e n k e p h a l i n and b e t a - e n d o r p h i n . W h e t h e r the i n c r e a s e d CSF M E - I R l e v e l s are due to an e n h a n c e d synthesis a n d r e l e a s e at spinal s i t e s or to a l o w e r t u r n o v e r of the p e p t i d e in C S F of o l d e r p a t i e n t s has to be a s c e r t a i n e d . The i n c r e a s e in C S F M E - I R m a y be r e l a t e d to the o b s e r v e d age-related changes in p a i n t h r e s h o l d and n a r c o t i c a n a l g e s i c effect which h a v e b e e n n o t i c e d in g e r i a t r i c p a t i e n t s (12,13).
References i.C. MISSALE, S. GOVONI, L. C A S T E L L E T T I , P . F . S P A N O and M. T R A B U C C H I , in: A . A g n o l i et Ai.Eds., A g i n g B r a i n and Ergot A l k a l o i d s , A g i n g Voi.23, R a v e n P r e s s pp: 15-22, (1983). 2. C. M I S S A L E , S. GOVONI, L. CASTELLETTI, P.F. S P A N O AND M. T R A B U C C H I , B r a i n Res. 262, 160-162, (1983). 3. A. DUPONT, P. SANARD, Y. MACAND, F. L A B R I E and J.K. BOISSIER, L i f e S c i e n c e s , 29, 2317-2322, (1981). 4. C. MISSALE, S. GOVONI, L. CROCE, A. BOSIO, P.F. S P A N O and M. T R A B U C C H I , J. N e u r o c h e m . , 40, 20-24, (1983). 5. O. HORNYKIEWICZ, Modification of cell to cell signals d u r i n g n o r m a l and p a t h o l o g i c a l aging. ( Eds S. G O V O N I and F. B A T T A I N I ) S p r i n g e r V e r l a g pp. 169-182, (1987). 6. T.D. H E X U M , I. H A N B A U E R , S. GOVONI, H.Y.T. YANG, and E.COSTA, N e u r o p e p t i d e s , l, 137-142, (1980). 7. S . G O V O N I , I. H A N B A U E R , T.D. HEXUM, H.Y.T. YANG, G.D. K E L L Y and E.COSTA, Neuropharmacology, 20, 639-645, (1981). 8. R . J . M I L L E R , R.H.CHANG, I. LEIGHTAN and P . Q U A T R E C A S A S , Life Sci., 22, 379-388, (1978). 9. S. GOVONI, G. PASINETTI, A. BIANCHI, M. GADOLA and M. T R A B U C C H I , A l c o h o l and D r u g Res., Z, 93-98, (1986).
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i0. S. GOVONI, G. PASINETTI, M.R. INZOLI, R. ROZZINI and M. TRABUCCHI, Life Sci., 35, 2594-2552, (1984). ii. F.W. BACH, K.JENSEN, N.BLEGUAD, M.FENGER, R . J O R D A L and J. OLESEN, Cephalalgia, 5, 23-27, (1983). 12. J . W . B E L V I L L E , W.H. FORREST, E. M I L L E R and B.W.BROWN, JAMA, 217, 1835-1841, (1971). 13. R.F.KAIKO, Clin. Pharmacol. Ther., 2_88, 832-826. (1980).