CSF Oxytocin Clinical
in Anorexia Nervosa and Pathophysiologic
and Bulimia Considerations
Nervosa:
Mark A. Demitrack, M.D., Michael D. Lesem, M.D, Sam J. Listwak, Harry A. Brandt, M.D., David C. Jimerson, M.D., and Philip W. Gold,
Oxytocin
is a hypothalamic
centrally
and
peripherally
f rom
experimental
pairs
consolidation
iors and tric
of
is released
oxytocin
level anorexia,
limia
of five
women
that
not
that
was
significantly
imbehav-
the
mean
women women lower
the
J
Psychiatry
1990;
bu-
level
of
147:882-886)
CNS
and
lateral
be
ventricles
routes
(3-7).
by
it exhibits
version
of the American 1988. Received
which
These
latter
oxytocin
a pronounced
path-
reaches
cincadian
the
rhythm
of a paper
presented
at the
Psychiatric Association, Aug. 11, 1989; revision
141st
Montreal,
annual
May
meeting
7-12,
accepted Dec. Neuroendocrinology Dr. Demitrack,
received Nov. 21, 1989; 21, 1989. From the Unit on Eating Disorders, Clinical Branch, NIMH. Address reprint requests to Unit on Eating Disorders, NIMH, Bldg. 10, Rm.
35-231,
Rockville
9000
The authors tocin antibody.
882
effects
its
role
the
thank
Pike, Dr.
Bethesda,
Delbert
Fisher
MD
20892.
for
providing
the
been
shown
effects
to
on
neuropeptides.
of
antioxy-
One
oxytocin
are
in reproduction
have
the
the
CNS
effects
mans
of vasopressin.
show
that
appear
For
oxytocin
kind
of
classically
nizing
vasopressin-induced
pituitary
nizes
the
axis
in hu-
release
of by
the
antago-
from
oxytocin
the
an-
antagonizes
consolidation
of
learning
(12, 13). Specifmarkedly antago-
consolidate
conditioned
anorexia
gerated, verse
to
to
studies
aversive conditioning administered atone
capacity
to
reciprocal
activation
ACTH of
is
aversivety
condi-
learning (12). of the principal theoretical stimuli for the study was this capacity of oxytocin to influence
aversively of
be
example,
modulates
promotion
during oxytocin
attributes
unrelated to
(1 1 ). In addition,
acquired ically,
these
(HPA)
tenor
tioned One present
of
functionally
and
hypothalamic-pituitary-adrenal
learning.
nervosa
and
perseverative effects
of
concern
food
A cardinal
bulimia
intake
symptom
nervosa
with and
is the
the
weight
exag-
potential gain
ad-
(14).
We
have previously shown that underweight patients with anorexia nervosa show abnormally high levels of centrally directed vasopressin in association with a profound defect in the osmoregulation of plasma vasopressin (15). We therefore speculated that tow levels of centrally directed oxytocin in patients with anorexia nervosa might be working in concert with high levels of
Revised
to
vasopressin’s
xytocin is a hypothalamic neuropeptide containing nine amino acids. It is structurally similar to the closely associated hypothalamic neuropeptide vasopressin, and like vasopressin, it is transported from the hypothalamus to the posterior pituitary for release into systemic circulation (1, 2). Additional centrally directed oxytocin and vasopressin pathways, which emanate from neuronat groups distinct from those which secrete into plasma, convey these peptides to disparate sites in brain (3). Some of the principal neuronal projections are directed to limbic structures, such as the hippocampus and amygdata, and many are in close contact with the CSF at such sites as the floor of the ways may CSF, where (8, 9).
also
attributed
O
third
has
integrative
consonant with its peripheral effects on partunition and lactation-namely, the initiation of a coordinated repertoire of complex maternal behaviors in the postpartum period that establishes a suitable environment for infant offspring (10). Notably, other prominent
1 1 control subjects. Restricting anorexic patients’ low CSF oxytocin levels may reflect their persistently low f ood intake, and this behavior may exacerbate their tendency for perseverative preoccupation with adverse consequences of food intake. (Am
oxytocin
CSF an-
with
than
(2),
tong-lasting,
re-
bulimic
While the principal rote of peripherally secreted oxytocin is to promote uterine contraction during partunition and milk let-down during the postpartum period
gaswith
12 underweight
or 35 normal-weight
nervosa,
Data
or experimental
report
underweight
but
both
oxytocin
conditioned
feeding
authors
with
pathways.
indicate
aversively
after
The
stricting
directed
animals
distension.
orexic
neuropeptide
B.S., M.D.
vasopressin,
further
impairing
the
extinction
of
aversivety conditioned learning. This hypothesis has potential implications for understanding the neurobiotogic substrates for the disturbances in eating behavion seen in patients with eating disorders. Finally, neciprocat abnormalities in vasopressin and oxytocin may
work
tion
of the
together
HPA
to
amplify
stress-induced
activa-
axis.
Am
J
Psychiatry
147:7,
July
1990
DEMITRACK,
METHOD Subjects The control subjects were 1 1 normal-weight women aged 21-35 years (mean±SD=25.S±4.3 years). Their mean±SD percentage of average body weight was 98.S%±9.0%. They were studied during the early fotlicutar phase of the menstrual cycle (days 1-7). Each was free of significant medical or psychiatric illness and had been drug-free for at least 4 weeks before the study. The anorexic patients were 20 women who met the DSM-III-R criteria for anorexia nenvosa; their age range was 16-33 years, and the mean±SD was 24.3± 5.2 years. These patients were classified into those without histories of binge-eating and vomiting (restnicting, N=7), those with histories of binge-eating and vomiting to enhance weight loss (butimic, N= 12), and one patient who reported a history of chronic vomiting without binge-eating to induce weight toss. Each patient had been drug-free for at least 6 weeks before the study. With the exception of their tow weights, these patients were nutritionally stable and without evidence of neunologic, renal, cardiovascular, or hepatic disease. Eighteen patients (five restricting, 12 butimic, one with chronic vomiting) were studied while chronically underweight, i.e., weighing less than 80% of average body weight (mean±SD=61.9%± 6.4%) for 6 months or more. Eighteen patients (seven restricting, 10 bulimic, one with chronic vomiting) were again studied at the end of refeeding. After maintaming their goat weights for 3 weeks, 16 patients (five restricting, 10 butimic, one with chronic vomiting) were again studied. The mean goal weight of these 16 patients was 84.7% ±2.0% of average body weight. Because there were logistical problems and some patients left before completing the program, only 14 patients were studied at all three phases of treatment. The patients with butimia nervosa were 35 women who met the DSM-III-R criteria for bulimia nervosa and ranged in age from 17 to 34 years (mean± SD=24.4±4.4 years). The average body weight for the group ranged from 85% to 1 10% of average body weight (mean=90.S% ±7.7%). Each was free of significant medical illness. These patients were studied within 72 hours of admission and after 3 weeks of voluntary abstinence from binge-eating and vomiting. Alt control subjects and patients were studied while hospitalized at the Unit on Eating Disorders at the Clinical Center of the National Institutes of Health and gave informed consent before participating in the studies.
Procedure The lumbar puncture was performed ject was lying on her left side, between a.m. after an overnight fast followed Thirty milliliters of CSF were withdrawn
Am
J
Psychiatry
I 47:7,
July
1990
while the sub9:00 and 9:30 by bed rest. over 15-30
LESEM,
LISTWAK,
ET AL.
minutes. A 1-mt aliquot representing the 1 ith of 30 ml was used for measurement of oxytocin. Oxytocin 1evets in CSF were determined by means of a nadioimmunoassay procedure involving an antioxytocin antibody, with modifications in both the extraction and assay procedure for adaptation to this laboratory (16). Briefly, CSF samples (1 ml each) were applied to C18 Sep-Paks that had been washed first with 10 ml of methanol, then with 10 ml of 0.1% tnifluoroacetic acid. Unabsorbed material was then washed off the Sep-Pak with 10 ml of 0.1% trifluoroacetic acid. The oxytocin was then eluted from the Sep-Pak with 6 ml of a solution of 60% acetonitnite and 40% 0.1% tnfluoroacetic acid (in water, volume for volume). This eluant was collected and the solvent removed by means of a Savant Vacuum Concentrator. The sample was then reconstituted in 0.5 ml of assay buffer (0.063 M sodium phosphate and 0.013 M sodium EDTA, pH 7.4, containing 0.02% sodium azide, 0.1% Triton X100, 250 kIU/ml apnotinin, and 1% normal rabbit serum) and allowed to sit for 1 hour at 4 #{176}C; 100 jiliter of the sample was then pipetted in duplicate for the radioimmunoassay. To each sample tube and to an oxytocin standard curve was added 100 p.liter of antiserum R-421 (final dilution of 1:40,000). The sampies were then incubated for 48 hours at 4 #{176}C, after which 100 .diter of HPLC-punified iodine-12S oxytocm tracer (approximately 3,000 counts/mm per tube) was added to each tube, and the incubation continued for another 24 hours at 4 #{176}C. At this time, a second antiserum (goat antirabbit gamma globulin) was added, and the incubation continued overnight (approximately 15 hours) to precipitate the bound counts. The samples were centrifuged to separate bound from free oxytocin, and the pellets were counted to determine bound counts/mm. The recovery of oxytocin added to hormone-free CSF was greater than 95%. The detection limit of this assay was 1.0 pg/mt. The intraand interassay coefficients of variation at specific oxytocin levels were as fottows: 6.09% and 10.40% at 3.60 pg/mi, 4.84% and 4.38% at 6.94 pg/mt, 2.64% and 8.35% at 13.90 pg/mi, and 3.91% and 7.08% at 31.50 pg/mi, respectively. Because of the small sample sizes, nonparametnic statistical measures were used for comparison of the data. Painwise comparisons were made with the MannWhitney U test, and comparison of group means was performed by means of Friedman’s test for multiple comparisons with modifications as described by Conover (17).
RESULTS The overall group of underweight anorexic patients and normal control subjects had similar CSF oxytocin levels (z1.60, p
in Anorexia Nervosa and Pathophysiologic
and Bulimia Considerations
Nervosa:
Mark A. Demitrack, M.D., Michael D. Lesem, M.D, Sam J. Listwak, Harry A. Brandt, M.D., David C. Jimerson, M.D., and Philip W. Gold,
Oxytocin
is a hypothalamic
centrally
and
peripherally
f rom
experimental
pairs
consolidation
iors and tric
of
is released
oxytocin
level anorexia,
limia
of five
women
that
not
that
was
significantly
imbehav-
the
mean
women women lower
the
J
Psychiatry
1990;
bu-
level
of
147:882-886)
CNS
and
lateral
be
ventricles
routes
(3-7).
by
it exhibits
version
of the American 1988. Received
which
These
latter
oxytocin
a pronounced
path-
reaches
cincadian
the
rhythm
of a paper
presented
at the
Psychiatric Association, Aug. 11, 1989; revision
141st
Montreal,
annual
May
meeting
7-12,
accepted Dec. Neuroendocrinology Dr. Demitrack,
received Nov. 21, 1989; 21, 1989. From the Unit on Eating Disorders, Clinical Branch, NIMH. Address reprint requests to Unit on Eating Disorders, NIMH, Bldg. 10, Rm.
35-231,
Rockville
9000
The authors tocin antibody.
882
effects
its
role
the
thank
Pike, Dr.
Bethesda,
Delbert
Fisher
MD
20892.
for
providing
the
been
shown
effects
to
on
neuropeptides.
of
antioxy-
One
oxytocin
are
in reproduction
have
the
the
CNS
effects
mans
of vasopressin.
show
that
appear
For
oxytocin
kind
of
classically
nizing
vasopressin-induced
pituitary
nizes
the
axis
in hu-
release
of by
the
antago-
from
oxytocin
the
an-
antagonizes
consolidation
of
learning
(12, 13). Specifmarkedly antago-
consolidate
conditioned
anorexia
gerated, verse
to
to
studies
aversive conditioning administered atone
capacity
to
reciprocal
activation
ACTH of
is
aversivety
condi-
learning (12). of the principal theoretical stimuli for the study was this capacity of oxytocin to influence
aversively of
be
example,
modulates
promotion
during oxytocin
attributes
unrelated to
(1 1 ). In addition,
acquired ically,
these
(HPA)
tenor
tioned One present
of
functionally
and
hypothalamic-pituitary-adrenal
learning.
nervosa
and
perseverative effects
of
concern
food
A cardinal
bulimia
intake
symptom
nervosa
with and
is the
the
weight
exag-
potential gain
ad-
(14).
We
have previously shown that underweight patients with anorexia nervosa show abnormally high levels of centrally directed vasopressin in association with a profound defect in the osmoregulation of plasma vasopressin (15). We therefore speculated that tow levels of centrally directed oxytocin in patients with anorexia nervosa might be working in concert with high levels of
Revised
to
vasopressin’s
xytocin is a hypothalamic neuropeptide containing nine amino acids. It is structurally similar to the closely associated hypothalamic neuropeptide vasopressin, and like vasopressin, it is transported from the hypothalamus to the posterior pituitary for release into systemic circulation (1, 2). Additional centrally directed oxytocin and vasopressin pathways, which emanate from neuronat groups distinct from those which secrete into plasma, convey these peptides to disparate sites in brain (3). Some of the principal neuronal projections are directed to limbic structures, such as the hippocampus and amygdata, and many are in close contact with the CSF at such sites as the floor of the ways may CSF, where (8, 9).
also
attributed
O
third
has
integrative
consonant with its peripheral effects on partunition and lactation-namely, the initiation of a coordinated repertoire of complex maternal behaviors in the postpartum period that establishes a suitable environment for infant offspring (10). Notably, other prominent
1 1 control subjects. Restricting anorexic patients’ low CSF oxytocin levels may reflect their persistently low f ood intake, and this behavior may exacerbate their tendency for perseverative preoccupation with adverse consequences of food intake. (Am
oxytocin
CSF an-
with
than
(2),
tong-lasting,
re-
bulimic
While the principal rote of peripherally secreted oxytocin is to promote uterine contraction during partunition and milk let-down during the postpartum period
gaswith
12 underweight
or 35 normal-weight
nervosa,
Data
or experimental
report
underweight
but
both
oxytocin
conditioned
feeding
authors
with
pathways.
indicate
aversively
after
The
stricting
directed
animals
distension.
orexic
neuropeptide
B.S., M.D.
vasopressin,
further
impairing
the
extinction
of
aversivety conditioned learning. This hypothesis has potential implications for understanding the neurobiotogic substrates for the disturbances in eating behavion seen in patients with eating disorders. Finally, neciprocat abnormalities in vasopressin and oxytocin may
work
tion
of the
together
HPA
to
amplify
stress-induced
activa-
axis.
Am
J
Psychiatry
147:7,
July
1990
DEMITRACK,
METHOD Subjects The control subjects were 1 1 normal-weight women aged 21-35 years (mean±SD=25.S±4.3 years). Their mean±SD percentage of average body weight was 98.S%±9.0%. They were studied during the early fotlicutar phase of the menstrual cycle (days 1-7). Each was free of significant medical or psychiatric illness and had been drug-free for at least 4 weeks before the study. The anorexic patients were 20 women who met the DSM-III-R criteria for anorexia nenvosa; their age range was 16-33 years, and the mean±SD was 24.3± 5.2 years. These patients were classified into those without histories of binge-eating and vomiting (restnicting, N=7), those with histories of binge-eating and vomiting to enhance weight loss (butimic, N= 12), and one patient who reported a history of chronic vomiting without binge-eating to induce weight toss. Each patient had been drug-free for at least 6 weeks before the study. With the exception of their tow weights, these patients were nutritionally stable and without evidence of neunologic, renal, cardiovascular, or hepatic disease. Eighteen patients (five restricting, 12 butimic, one with chronic vomiting) were studied while chronically underweight, i.e., weighing less than 80% of average body weight (mean±SD=61.9%± 6.4%) for 6 months or more. Eighteen patients (seven restricting, 10 bulimic, one with chronic vomiting) were again studied at the end of refeeding. After maintaming their goat weights for 3 weeks, 16 patients (five restricting, 10 butimic, one with chronic vomiting) were again studied. The mean goal weight of these 16 patients was 84.7% ±2.0% of average body weight. Because there were logistical problems and some patients left before completing the program, only 14 patients were studied at all three phases of treatment. The patients with butimia nervosa were 35 women who met the DSM-III-R criteria for bulimia nervosa and ranged in age from 17 to 34 years (mean± SD=24.4±4.4 years). The average body weight for the group ranged from 85% to 1 10% of average body weight (mean=90.S% ±7.7%). Each was free of significant medical illness. These patients were studied within 72 hours of admission and after 3 weeks of voluntary abstinence from binge-eating and vomiting. Alt control subjects and patients were studied while hospitalized at the Unit on Eating Disorders at the Clinical Center of the National Institutes of Health and gave informed consent before participating in the studies.
Procedure The lumbar puncture was performed ject was lying on her left side, between a.m. after an overnight fast followed Thirty milliliters of CSF were withdrawn
Am
J
Psychiatry
I 47:7,
July
1990
while the sub9:00 and 9:30 by bed rest. over 15-30
LESEM,
LISTWAK,
ET AL.
minutes. A 1-mt aliquot representing the 1 ith of 30 ml was used for measurement of oxytocin. Oxytocin 1evets in CSF were determined by means of a nadioimmunoassay procedure involving an antioxytocin antibody, with modifications in both the extraction and assay procedure for adaptation to this laboratory (16). Briefly, CSF samples (1 ml each) were applied to C18 Sep-Paks that had been washed first with 10 ml of methanol, then with 10 ml of 0.1% tnifluoroacetic acid. Unabsorbed material was then washed off the Sep-Pak with 10 ml of 0.1% trifluoroacetic acid. The oxytocin was then eluted from the Sep-Pak with 6 ml of a solution of 60% acetonitnite and 40% 0.1% tnfluoroacetic acid (in water, volume for volume). This eluant was collected and the solvent removed by means of a Savant Vacuum Concentrator. The sample was then reconstituted in 0.5 ml of assay buffer (0.063 M sodium phosphate and 0.013 M sodium EDTA, pH 7.4, containing 0.02% sodium azide, 0.1% Triton X100, 250 kIU/ml apnotinin, and 1% normal rabbit serum) and allowed to sit for 1 hour at 4 #{176}C; 100 jiliter of the sample was then pipetted in duplicate for the radioimmunoassay. To each sample tube and to an oxytocin standard curve was added 100 p.liter of antiserum R-421 (final dilution of 1:40,000). The sampies were then incubated for 48 hours at 4 #{176}C, after which 100 .diter of HPLC-punified iodine-12S oxytocm tracer (approximately 3,000 counts/mm per tube) was added to each tube, and the incubation continued for another 24 hours at 4 #{176}C. At this time, a second antiserum (goat antirabbit gamma globulin) was added, and the incubation continued overnight (approximately 15 hours) to precipitate the bound counts. The samples were centrifuged to separate bound from free oxytocin, and the pellets were counted to determine bound counts/mm. The recovery of oxytocin added to hormone-free CSF was greater than 95%. The detection limit of this assay was 1.0 pg/mt. The intraand interassay coefficients of variation at specific oxytocin levels were as fottows: 6.09% and 10.40% at 3.60 pg/mi, 4.84% and 4.38% at 6.94 pg/mt, 2.64% and 8.35% at 13.90 pg/mi, and 3.91% and 7.08% at 31.50 pg/mi, respectively. Because of the small sample sizes, nonparametnic statistical measures were used for comparison of the data. Painwise comparisons were made with the MannWhitney U test, and comparison of group means was performed by means of Friedman’s test for multiple comparisons with modifications as described by Conover (17).
RESULTS The overall group of underweight anorexic patients and normal control subjects had similar CSF oxytocin levels (z1.60, p
