Life Sciences, Vol. 31, pp. 1867-1870
Printed in the U.S.A.
Pergamon Press
PLASMA MEASURES OF B-ENDORPHIN-LIKE IMMUNOREACTIVITY
IN D E P R E S S I V E S A N D O T H E R P S Y C H I A T R I C S U B J E C T S
J. M a t t h e w s ,
H. Akil,
J. Greden,
a n d S. W a t s o n
Mental Health Research Institute
University of Michigan
Ann Arbor, M i c h i g a n 4 8 1 0 9
(Received in final form June 14, 1982)
Summary
We r e p o r t the u t i l i z a t i o n o f a v e r y
sensitive r a d i o i m m u n o a s s a y
for
plasma
B-END, w h i c h
h a s e n a b l e d us to e x a m i n e the s u p p r e s s i b i l i t y o f
B-END-like material, under
dexamethasone
challenge, on
a
population
of depressed patient~.
We h a v e found that B - E N D - l i k e m a t e r i a l in p l a s m a
is s u p p r e s s e d b y d e x a m e t h a s o n e in a
psychiatric
control population,
w h e r e a s s u p p r e s s i o n is less
l i k e l y in
endogenously
depressed
patients.
T h e r e also
a p p e a r s to b e a d i s s o c i a t i o n b e t w e e n
c o r t i s o l and B - E N D m e a s u r e s (tested at 4:00
PM)
in the e n d o g e n o u s l y d e p r e s s e d group.
S e v e r a l l i n e s o f e v i d e n c e d e m o n s t r a t e an a b n o r m a l i t y in the
hypothalmic-pituitary-adrenal
axis in p a t i e n t s
with
endogenous
d e p r e s s i o n (1,2,3).
T h e r e are m u l t i p l e l e v e l s w h e r e the d e f e c t
might be localized.
The d e x a m e t h a s o n e
suppression
test
(DST)
m e a s u r e s the
pituitary-adrenal
interface.
This p a p e r a t t e m p t s
to m o v e t h e s t u d y o f the
endocrinology
of
depression
to
the
level o f
the
pituitary
and b r a i n .
Thus, we are i n t e r e s t e d in
m e a s u r i n g b e t a - e n d o r p h i n s i n c e it is p a r t o f the same
precursor
molecule, pro-opiomelanocortin,
as
A C T H (4,5).
We h a v e used a
d e x a m e t h a s o n e c h a l l e n g e to b e g i n to look at
the
regulation
of
the p i t u i t a r y
pro-opiomelanocortin
system.
Although one study
r e p o r t s no s u p p r e s s i o n w i t h d e x a m e t h a s o n e in normals, t h e p l a s m a
r a d i o i m m u n o a s s a y is d i f f i c u l t and m a y lead
to
false
negatives
(6).
We
have
therefore
addressed
here
the
following
two
questions:
a)
Does
dexamethasone
suppress
B-END
in
human
plasma?
and
b)
Is the p a t t e r n o f B - E N D s u p p r e s s i o n a l t e r e d in
endogenous depression?
Methods
B l o o d c o l l e c t i o n was c a r r i e d
out
via
angiocatheter
into
chilled EDnA
containing
vacutainers.
The s a m p l e s w e r e c e n t r i fuged at 4vC for I0 m i n u t e s and w e r e t h e n a l i q u o t e d into p l a s t i c
s t o r a g e v i a l s and a c i d i f i e d to a p H of 2.0 w i t h
1 N
HCI.
The
acldlfled serum
was
s t o r e d at -70 C u n t l l e x t r a c t l o n .
We used
Sep Pak C ~
c a r t r i d g e s (Waters)
for
~ur
extraction
procedure
which progided
90%
recovery
with
H - - B - E N D s p i k e d h u m a n serum
(9).
0024-3205/82/161867-04503.00/0
Copyright (c) 1982 Pergamon Press Ltd.
•
•
•
O
•
•
1868
8-endorphin in Depression
Vol. 31, No.s 16 & 17, 1982
The r a d i o i m m u n o a s s a y was c a r r i e d out in 150 m M Na p h o s p h a t e
b u f f e r w i t h 1% h u m a n serum a l b u m i n at p H
8.2
under
disequilibrium incubation.
The
a n t i s e r u m (rabbit Brenda) was used at a
final c o n c e n t r a t i o n o f 1 : 4 0 , 0 0 0 and s e p a r a t i o n was
carried
out
by
immunoprecipitation
with
goat
antirabbit
IgG.
Crossreactivity studies
with
our
antibody
showed
100%
crossreactivity with
B-LPH
and
N - a c e t y l - B - E N D , 50% w i t h B-END1_97,
and 30% w i t h N - a c e t y l - B - E N D q ..,
whereas
there
is
no
c~o~sreactivity
with
alpha-EN~?Zlgamma-END,
B-END?_~,
B-ENDg_I7,
m e t h i o n i n e e n k e p h a l i n , or a l p h a - m e l a n o c y t e - s t i m u ~ a ~ i n g
hormone.
The a s s a y is c a p a b l e o f m e a s u r i n g <1.5 f m o l e s / a s s a y t u b e (normal
resting
levels
of
B-END-like
immunoreactivity
are
2-5
fmoles/ml).
The v a l i d a t i o n o f the a s s a y and the
chromatography
o f the m a t e r i a l b e i n g m e a s u r e d h a v e b e e n d e s c r i b e d (9).
U s i n g this p r o c e d u r e we s t u d i e d a m i x e d p s y c h i a t r i c c o n t r o l
p o p u l a t i o n (N=5) v e r s u s a g r o u p o f e n d o g e n o u s d e p r e s s i v e s (N=6).
Under dexamethasone challenge serum
cortisol
and
serum
B-END
levels were
measured.
An
18 gauge a n g i o c a t h e t e r was i n s e r t e d
into an a n t e c u b i t a l arm v e i n at 1:30 PM;
20 ml
blood
samples
were obtained
at
20 m i n u t e i n t e r v a l s b e t w e e n 3:30 and 4:30 PM.
All s u b j e c t s w e r e t h e n g i v e n 1 mg of d e x a m e t h a s o n e at
11:30
PM
that evening
and
the
catheter
study
was
carried
out in an
i d e n t i c a l f a s h i o n t h e f o l l o w i n g day.
Both
cortisol
and
B-END
m e a s u r e s are
reported
as
the
means
of
the four t i m e p o i n t s
b e t w e e n 3:30 and 4:30 PM.
Thus:
I) s i m u l t a n e o u s
cortisol
and
B-END measures
were
obtained;
2)
subjects
were
their
own
controls;
a n d 3) r e p e a t e d m e a s u r e s e n a b l e
us
to
control
for
stress and
p u l s a t i l e r e l e a s e o f B-END.
The 4:00 PM t i m e p e r i o d
was c h o s e n
since
this
is
the
time
when
the
dexamethasone
suppression test
is m o s t
s e n s i t i v e in s e p a r a t i n g e n d o g e n o u s l y
d e p r e s s e d p a t i e n t s from normal i n d i v i d u a l s .
Results
T a b l e 1 shows t h e results.
The n o n - e n d o g e n o u s
psychiatric
c o n t r o l g r o u p showed a m e a n s u p p r e s s i o n o f t h e i r B - E N D l e v e l s o f
45% + 8 as
compared
to
18% + 8 s u p p r e s s i o n for the e n d o g e n o u s
depressives.
The m e a n H a m i l t o n r a t i n g for
depression
for
the
control
group
was
6 + 2 and
24 + 1
for
the
endogenous
depressives.
Further, t ~ e r e is an a b s e n c e
of
overlap
between
pre- and p o s t - d e x a m e t h a s o n e B - E N D l e v e l s in the c o n t r o l s , w h e r e as t h e r e
was
considerable
overlap
in e n d o g e n o u s d e p r e s s i v e s .
The p o s t - d e x a m e t h a s o n e B - E N D v a l u e s w e r e v e r y c l o s e l y
clustered
in the
controls
in c o n t r a s t to m u c h g r e a t e r v a r i a b i l i t y in the
endogenous depressives.
Since endogenously
depressed
patients
usually
exhibit
cortisal values
of
g r e a t e r t h a n 5.0 ug/dl a f t e r d e x a m e t h a s o n e ,
we h a v e r e v i e w e d
the
B-END
data
for
a
similar
cutoff.
A
conservative preliminary
post-dexamethasone
v a l u e o f 2.5 fm/ml
was a b o v e all n o n - e n d o g e n o u s p o s t - d e x a m e t h a s o n e v a l u e s .
As s u m m a r i z e d b y T a b l e 2,
B-END
levels
above
2.5
fm/ml
i d e n t i f i e d five
out
o f the six e n d o g e n o u s d e p r e s s i v e s , w h e r e a s
post-dexamethasone cortisol levels identified only
two
of
the
six e n d o g e n o u s
depressives.
Note t h a t t h e r e is g o o d a g r e e m e n t
for the a b s e n c e o f a b n o r m a l B - E N D and c o r t i s o l r e s p o n s e s in
the
non-endogenous
groups,
whereas
the
agreement
for
abnormal
Vol. 31, No.s 16 & 17, 1982
B-endorphin in Depression
Table
Non-endogenous
Subject
1
2
3
4
5
X + S.E.
BE P l a s m a
Pre d e x
6.8
3.7
2.6
2.8
2.4
+
T
+
•
T
0.8
0.4
0.2
1.2
0.3
3.5 + 0.7
level (fm/ml)
P o s t dex
+
T
+
;
•
6
7
8
9
i0
ii
X + S.E.
X BE P l a s m a
Pre d e x
3.6
3.3
10.2
1.8
5.4
3.9
+
•
•
•
•
•
0.4
0.8
1.6
0.3
0.9
1.2
4.7 + 1.3
0.3
0.3
0.2
0.3
0.i
+
T
•
•
•
•
45% + 8
0.8
1.7
1.8
0.2
0.5
0.9
3.8 + 0.7
Endogenous
#2
#3
#4
#5
6 + 2
#6
C+)
#7
#8
#9
#10
#ii
(+)
(+)
(-)
(+)
(+)
0%
4%
43%
32%
31%
0%
21
22
38
21
18
24
24 + 1
2
(-)
(-)
(-)
(-)
(-)
Suppression
Hamilton
Depression
Ratings
18% + 8
A b n o r m a l BE
Suppression
>2.5 fm/ml
#i
i0
1
7
8
-
Depressives
Table
Nonendogenous
Hamil ton
Depression
Ratin@s
70%
51%
23%
46%
33%
level (fm/ml)
P o s t dex
5.2
3.2
5.8
1.2
3.8
4.0
Controls
Suppression
.... 1.7 + 0 . i
Endogenous
Subject
1
Psychiatric
1.8
1.8
2.0
1.5
1.6
1869
Abnormal Cortisol
Suppression
>5.0 ug/dl
(-)
(-)
0
~
5
6
(-)
(-)
(-)
(-)
(-)
(-)
(+)
(+)
(-)
0
2
r e s p o n s e s is
poor
in
the
endogenously
depressed
group.
s h o u l d b e noted, h o w e v e r , t h a t
the
combination
of
B-END
cortisol measures
around
the 4 : 0 0 PM t i m e p o i n t i d e n t i f i e s
of the endogenously depressed patients.
It
and
all
1870
B-endorphln in Depression
Vol. 31, No.s 16 & 17, 1982
Discussion
T h e s e r e s u l t s s u g g e s t t h a t B - E N D - l i k e m a t e r i a l in p l a s m a is
suppressed by
dexamethasone
in
a non-endogenous
psychiatric
population, whereas
suppression
is less l i k e l y in e n d o g e n o u s l y
depressed patients.
Of i n t e r e s t is
the
observation
that
the
combination of
b o t h c o r t i s o l and B - E N D - l i k e m e a s u r e s i d e n t i f i e s
all o f the e n d o g e n o u s l y d e p r e s s e d
patients.
The
dissociation
between cortisol
and
B-END-like
values
in
the
endogenously
depressed patients warrants further
investigation
in
view
of
r e p o r t s s u g g e s t i n g that B - E N D and A C T H are s e c r e t e d in e q u i m o l a r
a m o u n t s (7).
S i m u l t a n e o u s m e a s u r e s o f ACTH, B-END, and c o r t i s o l
may help
explain
this
lack
of
agreement
and
possibly help
l o c a l i z e t h e defect.
This d i s c r e p a n c y m a y a l s o
be
related
to
the 3:30
to
4 : 3 0 PM s a m p l i n g t i m e s i n c e B - E N D and c o r t i s o l are
r e g u l a t e d d i f f e r e n t l y and h a v e d i f f e r e n t h a l f lives (8).
There
may be
a time
p e r i o d at w h i c h c o r t i s o l and B - E N D m e a s u r e s are
m o r e t i g h t l y linked.
Thus, t h e b e s t t i m e p o i n t
post-dexamethasone B - E N D m e a s u r e m e n t n e e d s to b e d e t e r m i n e d .
Twenty-four hour
circadian
rhythm
studies
are
thus
imperative.
The
large
d i f f e r e n c e s in the H a m i l t o n r a t i n g s in the p s y c h i a t r i c
controls
versus the
endogenous
depressives
suggests
that
severity of
i l l n e s m a y a c c o u n t for the lack o f s u p p r e s s i o n in the e n d o g e n o u s
depressives;
thus, future s t u d i e s
need
to
control
for
this
variable.
Studies
on
normal
c o n t r o l s and sex d i f f e r e n c e s are
n o w b e i n g c a r r i e d out.
Acknowledgements
T h i s w o r k was
supported
by NIMH Grant
#R01-MH36168.
References
i.
2
3
4
5
6
7
8.
9.
J.L. GIBBONS, Arch. Gen. P s y c h i a t r y i0 5 7 2 - 5 7 5 (1964)
B.J. C A R R O L L and
B.M. DAVIES,
Brit. Med. J.
1
789-791
(1970)
B.J. CARROLL, G.C. C U R T I S and J. MENDELS, Arch. Gen. Psych.
33 1 0 5 1 - 1 0 5 8 (1976)
B. E I P P E R and
R. MAINS,
J. S u p r e m o l e c u l a r
Structure
8
2 4 7 - 2 6 2 (1978)
S. NAKANISHI, A. INOUE, T. KITA, M. NAKAMURA, A.C.Y. CHANG,
S.N. C O H E N and S. NUMA, N a t u r e 278 4 2 3 - 4 2 7 (1979)
N.H. KAHIN,
S.C. RISCH,
R.M. COHEN,
T. I N S E L
and
D.L. MURPHY, S c i e n c e 209 8 2 7 - 8 2 8 (1980)
R. GUILLEMIN, T. VARGO,
J. ROSSIER,
S. MINICK,
N. LING,
C. RIVIER, W. VALE
and
F. BLOOM,
Science
197
1357-1360
(1977)
M. ENDO,
J. ENDO,
M. N I S H I K U B O ,
T. Y A M A G U C H I
and
N. HATONANI, P s y c h o n e u r o e n d o c r i n o l o g y
Workshop
Conf. Int.
Soc. P s y c h o n e u r o e n d o c r i n o l o g y ,
Mieken
(Karger,
Basel),
22-31 (1974)
C. CAHILL, J. M A T T H E W S and H. AKIL, (submitted)