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.
doi:10.1016/j.psyneuen.2004.02.003 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.
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