Psychoneuroendocrinology (2004) 29, 1341–1344
Effects of PhD examination stress onallopregnanolone and cortisol plasma levels andperipheral benzodiazepine receptor density
Hal A. Droogleever Fortuyna, Frank van Broekhovena,*, Paul N. Spanb,Torbjo
¨mc, Frans G. Zitmana,1, Robbert J. Verkesa
aUnit for Clinical Psychopharmacology and Neuropsychiatry, 331 Department of Psychiatry,University Medical Center Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, The NetherlandsbDepartment of Chemical Endocrinology, University Medical Center Nijmegen,Nijmegen, The NetherlandscDepartment of Clinical Sciences, Obstetrics and Gynecology, Umea
Received 8 September 2003; received in revised form 21 January 2004; accepted 17 February 2004
Summary Peripheral benzodiazepine receptor (PBR) density in blood platelets and
plasma allopregnanolone concentration in humans were determined following acute
stress as represented by PhD examination. Fifteen healthy PhD students partici-
pated. Heart rate, blood pressure, plasma allopregnanolone, plasma cortisol, and
PBR density were measured at different time points.
Allopregnanolone and cortisol concentration and PBR density were significantly
increased during examination. A positive correlation between allopregnanolone and
# 2004 Elsevier Ltd. All rights reserved.
chondrial membrane, plays a role in the translo-cation of cholesterol from the outer to the inner
The peripheral benzodiazepine receptor (PBR),
which is located in the central and peripheral ner-
vous system, is involved in steroidogenesis and is
considered to be an important potential thera-
metabolite of progesterone and a potent positive
allosteric modulator of the gamma-aminobutyricacid
*Corresponding author. Tel.: +31-24-3610966; fax: +31-24-
E-mail address: [email protected]
(F. van Broe-
effect appears to depend on the presence or
Current address: Department of Psychiatry, Leiden Univer-
sity Medical Center, Leiden, The Netherlands.
0306-4530/$ - see front matter # 2004 Elsevier Ltd. All rights reserved.
As allopregnanolone can both accumulate in the
description of the study to the subjects, written
brain irrespective of supply from peripheral endo-
In brief, PhD examination in the Netherlands
synthesised de novo from sterol precursors, it is
constitutes the presentation and defense of one’s
scientific work in front of a board of professors.
rosteroids are involved in many central nervous
After the presentation and defense, is a break of
system disorders like depression, anxiety, learningand
15 min in which the board retires and reaches a
conclusion about the PhD student as having passed
In animals, brain and plasma allopregnano-
the examination or not. After the break, the
lone levels, and in animals and humans, platelet
board returns and informs the PhD student about
PBR density, both increase following acute stress
its conclusion. PhD examination is considered to
be a stressful event and was therefore used in the
data exist with respect to plasma allopregnano-
present study as a model of acute stress
lone levels following acute stress in humans
cortisol plasma concentration, blood pressure
thermore, the relation between PBR density and
(BP), and heart rate (HR). Plasma levels of allo-
allopregnanolone plasma levels following acutestress has not been investigated. The mechanism
pregnanolone, and the maximal number of binding
by which PBR density rapidly increases following
sites in blood platelets (Bmax) for the PBR specific
acute stress is not known. It could be that there
ligand 3H-PK11195 were measured. All measure-
exist PBR containing vesicles that are released by
ments took place four weeks before the PhD
fusion of the vesicles with the cell membrane. If
examination (T1), 45 min before the examination
this is true and how acute stress causes this exo-
(T2), during the examination (T3), and four weeks
after the graduation (T4). Blood samples at T2
In the present study, we tried to replicate pre-
were drawn between 12:30 and 15:00 h; blood
vious findings of increased PBR density following
samples at T3 were drawn at 14:30 or 16:30 h.
acute stress as represented by an examination
The preparation of platelets was essentially as
examined our hypothesis that in acute stress,
resulting blood platelet fractions were used for
plasma allopregnanolone concentration increases
the PBR assay, the (plasma) supernatant was used
and that this increase is correlated with the
for the hormone measurements. Both were kept
increase in PBR density. Recently, it has been
at À80 C until analysis. The PBR binding assay
shown that allopreganolone levels decrease during
was performed essentially as described earlier
panic provocation in patients with panic disorder
Separation of bound from free 3H-PK11195, was
induced panic attack, patients with panic disorder
measured by radioimmunoassay as described in
fail to maintain compensatory increased allo-
pregnanolone levels. Considering the anxiolytic
levels were measured by an immunofluorometric
effect of allopregnanolone and the important role
assay (IFMA), the time-resolved fluoroimmunoassay
of PBR agonists in the biosynthesis of allopregna-
technique (Delfia) using the reagent kit 1244-060
nolone, a positive correlation between the two
DELFIA1 Cortisol Kit (Wallac OY, Turku, Finland)
during acute stress may support the suggestion
according to the manufacturer’s instructions.
that synthetic PBR agonists may prove to be
potent and effective new anxiolytic drugs.
used as controls in the Delfia assay. The intraassay coefficient of variation in the cortisolassay was 5% and inter assay 4.5%. The analytical
sensitivity was 15 nmol/l serum. The inter assaycoefficient of variation for the allopregnanolone
Fifteen healthy PhD students (12 men and 3
assay was 8.5% and the detection limit 18 pg.
women), with a mean age of 35 years (range
In the three women, all measurements took
29–41 years), were recruited by means of advertise-
place in the follicular phase except for one
ments. No abnormalities were found during physi-
measurement: baseline measurement (time point 1)
cal and psychiatric examination. After complete
in one woman took place in the luteal phase. As
a (physiologically) higher allopregnanolone value
was found at this time point compared to the
(F ¼ 12:38, df ¼ 3; 41, p < 0:001) with T3 > T1; T4.
other time points (time points 2–4) in this woman,
Bmax was significantly correlated with allo-
the difference in phase has not confounded the
pregnanolone plasma concentrations (r ¼ 0:35;
results of this study (high allopregnanolone levels
B ¼ 3:3 Â 10À5; F ¼ 5:92; df ¼ 1; 44; p ¼ 0:02).
were found during the PhD examination (time
Cortisol plasma concentrations were significantly
Differences in means at the different time
correlated with allopregnanolone plasma con-
points and regression coefficients and their corre-
centrations (r ¼ 0:40; B ¼ 598; F ¼ 7:97; df ¼
sponding Pearson correlation coefficients were
statistically tested using mixed model analyses of
F ¼ 8:08; df ¼ 1; 43; p ¼ 0:007) and diastolic BP
variance (ANOVA) with subjects as random factor
(r ¼ 0:38; B ¼ 18; F ¼ 6:89; df ¼ 1; 43; p ¼ 0:012).
and time points as fixed factor. Post-hoc compar-isons between time points were tested with pairedt-tests. In order to adjust for time of blood sam-pling at T
1 and T4, all samples from all time points
(T1–T4) were divided into two groups: samples
The results showed that allopregnanolone in
humans is increased following acute stress. Fur-thermore, this is the first study to show that, inacute psychological stress, the increase in plasma
allopregnanolone concentration is correlated withan increase in PBR density in blood platelets. The
Results of Bmax of PBR binding, allopregnanolone
fact that during the PhD examination, plasma cor-
and cortisol are presented in . No effect of
tisol concentration, BP, and HR reached their
time of blood sampling could explain the signifi-
highest scores, supported our assumption that PhD
cant differences in allopregnanolone, cortisol con-
examination is a valid model for acute stress.
The increase in PBR density during the examin-
There was a statistically significant effect of
ation in the present study is in accordance with
time for HR (F ¼ 17:6, df ¼ 3; 41, p < 0:001) with
previous findings. Karp and colleagues have shown
T3 > T1; T4; systolic BP (F ¼ 28:53, df ¼ 3; 41,
increased PBR densities in residents in psychiatry
PBR density in blood platelets and plasma allopregnanolone and cortisol levels. Bars indicate means and
error bars one standard deviation. Time points on the x-axis (1–4) refer to: four weeks before PhD examination (1);45 min before PhD examination (2); during PhD examination (3); and four weeks after PhD graduation (4). Bmax(maximal number of binding sites in blood platelets for the PBR specific ligand 3H-PK11195) expressed in 10À11 mol/mg protein, allopregnanolone in nmol/l, and cortisol in lmol/l. Significant effect of time (?) for PBR density(F ¼ 2:94, df ¼ 3; 42, p < 0:05) with T3 > T1; T2; plasma allopregnanolone (F ¼ 6:38, df ¼ 3; 42, p < 0:01) withT3 > T1; T2; T4; and plasma cortisol (F ¼ 5:27, df ¼ 3; 42, p < 0:01) with T3 > T1; T4.
immediately after the acute stress of board exam-
platelets of neuroleptic-treated schizophrenics. Eur. J.
The increase in plasma cortisol during acute
Gavish, M., Weizman, A., Karp, L., Tyano, S., Tanne, Z.,
1986b. Decreased peripheral benzodiazepine binding sites in
stress was not correlated with the increase in PBR
platelets of neuroleptic-treated schizophrenics. Eur. J.
density. This suggests that acute stress-evoked
increases in cortisol concentrations do not depend
Girdler, S.S., Straneva, P.A., Light, K.C., Pedersen, C.A., Mor-
row, A.L., 2001. Allopregnanolone levels and reactivity to
Considering the correlational nature of this
mental stress in premenstrual dysphoric disorder. Biol. Psy-chiatr. 49, 788–797.
study, no causal relationship between the increase
Karp, L., Weizman, A., Tyano, S., Gavish, M., 1989. Examin-
in PBR density and allopregnanolone concen-
ation stress, platelet peripheral benzodiazepine binding
tration can be drawn from the results. Further
sites, and plasma hormone levels. Life Sci. 44, 1077–1082.
research is needed to demonstrate if synthetic
Krueger, K.E., Papadopoulos, V., 1990. Peripheral-type benzo-
PBR agonists enhance the biosynthesis of allo-
diazepine receptors mediate translocation of cholesterolfrom outer to inner mitochondrial membranes in adrenocor-
pregnanolone and, subsequently, if they may be
tical cells. J. Biol. Chem. 265, 15015–15022.
of use in the treatment of anxiety disorders.
Krueger, K.E., Papadopoulos, V., 1992. Mitochondrial benzodia-
zepine receptors and the regulation of steroid biosynthesis.
Annu. Rev. Pharmacol. Toxicol. 32, 211–237.
Laconi, M.R., Casteller, G., Gargiulo, P.A., Bregonzio, C., Cab-
Altemus, M., Redwine, L.S., Leong, Y.M., Frye, C.A., Porges,
rera, R.J., 2001. The anxiolytic effect of allopregnanolone
S.W., Carter, C.S., 2001. Responses to laboratory psychoso-
is associated with gonadal hormonal status in female rats.
cial stress in postpartum women. Psychosom. Med. 63,
Majewska, M.D., 1992. Neurosteroids: endogenous bimodal
Ansseau, M., von Frenckell, R., Cerfontaine, J.L., Papart, P.,
modulators of the GABAA receptor. Mechanism of action
1991. Pilot study of PK 11195, a selective ligand for the per-
ipheral-type benzodiazepine binding sites, in inpatients
Majewska, M.D., Harrison, N.L., Schwartz, R.D., Barker, J.L.,
Paul, S.M., 1986. Steroid hormone metabolites are barbitu-
Baulieu, E.E., Robel, P., Schumacher, M., 1999. Neurosteroids:
rate-like modulators of the GABA receptor. Science 232,
from definition and biochemistry to physiopathologic func-
tion. In: Baulieu, E.E., Robel, P., Schumacher, M. (Eds.),
Mellon, S.H., 1994. Neurosteroids: biochemistry, modes of
Neurosteroids: A New Regulatory Function in The Nervous
action, and clinical relevance. J. Clin. Endocrinol. Metab.
System. Humana Press, Totowa, NJ, pp. 1–25.
Benraad, T.J., Foekens, J.A., 1990. Hydroxyapatite assay to
Purdy, R.H., Morrow, A.L., Moore, Jr., P.H., Paul, S.M., 1991.
measure epidermal growth factor receptor in human pri-
Stress-induced elevations of gamma-aminobutyric acid type
mary breast tumors. Ann. Clin. Biochem. 27, 272–273.
A receptor-active steroids in the rat brain. Proc. Natl.
Bicikova, M., Tallova, J., Hill, M., Krausova, Z., Hampl, R.,
2000. Serum concentrations of some neuroactive steroids in
Rupprecht, R., Holsboer, F., 1999. Neuroactive steroids:
women suffering from mixed anxiety-depressive disorder.
mechanisms of action and neuropsychopharmacological per-
spectives. Trends Neurosci. 22, 410–416.
Bixo, M., Andersson, A., Winblad, B., Purdy, R.H., Ba
¨hle, A., Romeo, E., di Michele, F., Pasini, A., Yassouridis,
T., 1997. Progesterone, 5alpha-pregnane-3,20-dione and
A., Holsboer, F., Rupprecht, R., 2002. GABA(A) receptor-
3alpha-hydroxy-5alpha-pregnane-20-one in specific regions
modulating neuroactive steroid composition in patients with
of the human female brain in different endocrine states.
panic disorder before and during paroxetine treatment. Am.
Brambilla, F., Biggio, G., Pisu, M.G., Bellodi, L., Perna, G.,
¨hle, A., Romeo, E., di Michele, F., Pasini, A., Hermann, B.,
Bogdanovich-Djukic, V., Purdy, R.H., Serra, M., 2003. Neu-
Gajewsky, G., Holsboer, F., Rupprecht, R., 2003. Induced
rosteroid secretion in panic disorder. Psychiatr. Res. 118,
panic attacks shift gamma-aminobutyric acid type A recep-
tor modulatory neuroactive steroid composition in patients
Drugan, R.C., 1996. Peripheral benzodiazepine receptors: mol-
with panic disorder: preliminary results. Arch. Gen. Psy-
ecular pharmacology to possible physiological significance
in stress-induced hypertension. Clin. Neuropharmacol. 19,
van Rood, Y., Goulmy, E., Blokland, E., Pool, J., van Rood, J.,
van Houwelingen, H., 1991. Month-related variability in
Gavish, M., Weizman, A., Karp, L., Tyano, S., Tanne, Z.,
immunological test results; implications for immunological
1986a. Decreased peripheral benzodiazepine binding sites in
follow-up studies. Clin. Exp. Immunol. 86, 349–354.
Vox Sanguinis (2006) 90 , 77–84 Malaria and blood transfusion A. D. Kitchen1 & P. L. Chiodini2 1 National Blood Service, London, UK 2 The Hospital for Tropical Diseases, London, UK The transmission of malaria by blood transfusion was one of the first recorded inci-dents of transfusion-transmitted infection. Although a number of different infectionshav
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