Plasma β-endorphin, pain thresholds and anxiety levels across the human menstrual cycle

Physiology & Behavior, Vol. 32, pp. 31-34. Copyright ~ Pergamon Press Ltd., 1984. Printed in the U.S.A.

0031-9384/84 $3.00 + .00

Plasma/3-Endorphin, Pain Thresholds and Anxiety Levels Across the Human Menstrual Cycle J A N E L . V E I T H , 1 J U L I E A N D E R S O N , S U E A. S L A D E , P A M E L A T H O M P S O N , G A R Y R. L A U G E L A N D S H E L L Y G E T Z L A F W a s h i n g t o n S t a t e U n i v e r s i t y , P u l l m a n , W A 99164 R e c e i v e d 17 M a r c h 1983 VEITH, J. L., J. ANDERSON, S. A. SLADE, P. THOMPSON, G. R. LAUGEL AND S. GETZLAF. Plasma [3-endorphin, pain thresholds and anxiety levels across the human menstrual cycle. PHYSIOL BEHAV 32(1) 31-34, 1984.--Nine normally cycling women and seven other women employing oral contraception were tested during five phases (menstrual, follicular, ovulatory, luteal and premenstrual) of their menstrual cycle. The procedure consisted of administration of an anxiety inventory and determination of pain detection and pain thresholds in response to electric shock and the cold pressor task. Venipunctures were also performed and the plasma of normally menstruating women later assayed for/3-endorphin. Analyses revealed that the variance but not the mean levels in peripheral/3-endorphin levels significantly differed (p<0.01) across the menstrual cycle with the greatest amount of variance found during the ovulatory phase and the least during the luteal phase. The high variance during the period around ovulation was due to several subjects having extremely elevated B-endorphin levels which possibly may have resulted from the occurrence of ovulation. Furthermore, a significant positive correlation between anxiety levels and/3-endorphin levels was found only during the menstrual phase. The absence of findings concerning cyclic variation in pain thresholds is contrary to earlier reports and indicates that such a phenomenon may be dependent upon the paradigm employed. /3-Endorphin

Menstrual cycle

Pain thresholds

Anxiety

A small but conflicting literature exists concerning the phenomenon of human menstrual cycle related changes in pain thresholds. Herren [7], employing a two point threshold paradigm for touch and pain, reported that premenstrual thresholds in normal cycling women were significantly lower (more sensitive) than those of either the postmenstrual or intermenstrual phases. A later study [16], examining pain tolerance in response to prolonged electric shock indicated that greatest sensitivity occurred during the menstrual phase. In contrast, others [5,12], using the same population, have found heightened sensitivity to pain in response to radiant heat during the ovulatory phase. It may be conjectured that the lack of consistency in findings among these reports may be a function of the pain stimulus employed (radiant heat, tactile pressure, electric shock) and/or the type of response examined (pain threshold, pain tolerance). Both variables, stimulus and response type, have been demonstrated to contribute variance to the obtained results [2,15]. At present, the physiological mechanism(s) underlying cyclic variation in pain thresholds is unknown. While behavioral change related to the menstrual cycle is typically attributed to fluctuations in the hypothalamic-pituitarygonadal (HPG) axis [5,12], the endorphins [1, 8, 10] may also be implicated on the basis of the strong evidence concerning

their capacity to induce analgesia across a number of species, including humans [11, 18, 21]. No reports are presently available conceming human menstrual cycle-related fluctuations in endorphin levels. However, evidence exists indicating that the endorphin system may interact with the HPG axis. In women, the endorphins appear to have the capacity to play an inhibitory role in L H regulation during high estrogen and estrogenprogesterone phases [13]. Furthermore,/3-endorphin levels in the hypophyseal portal bloOd of rhesus and pigtailed monkeys peaked during the mid-to-late follicular and luteal phases but were undetectable during the mertstrual phase [20]. However, brain levels offl-endorphin in female rats did not vary cyclically [19]. In addition, human plasma levels of ~-endorphin have been demonstrated to increase during pregnancy [4] and decline following menopause [3], leading some to speculate that variation in this neuropeptide category may be intricately tied to reproduction related mood changes [6]. On the basis of this evidence, it may be proposed that plasma /3-endorphin systematically fluctuates across the human menstrual cycle. Furthermore, this variation in peptide levels may be related to cyclic variations in pain thresholds.

1Requests for reprints should be addressed to Jane Veith, Ph.D., Department of Psychology-4830, Washington State University, Pullman, WA 99164.

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VI~I'I'H t;1 AI METHOD

Participants Volunteers were recruited by posted advertisement and word of mouth. During the participant recruitment stage, participants were informed that the study's aim was to examine the relationship between /3-endorphin and pain thresholds across five phases of the menstrual cycle. Participants consisted of two groups: Group 1 which was comprised of nine normally menstruating women who had not employed oral contraceptives for at least six months prior to the initiation of the study and Group 2 which consisted of seven women who were presently using oral contraception. The mean age of the Group 1 participants was 25.67 years old as compared to the mean age of Group 2 which was 24.71; this difference was not significant as determined by a t-test. All subjects were in good health and with the exception of one, not employing prescription medication other than oral contraceptives. Participants were instructed to refrain from use of alcohol, pain medication, and other drugs 24 hours preceding each test session. At the termination of the study, each received ten dollars for her participation. Procedure A month preceding behavioral testing, all women were instructed to maintain basal body temperature (BBT) menstrual calendars as a method of determining both cycle regularity and occurrence of ovulation. During the subsequent month, participants were instructed to continue recording their BBT. Each participant was tested at five phases of her menstrual cycle: menstrual (days 2-4), follicular (days 8-10), ovulatory (as determined from BBT chart using a three point criteria [9]), luteal (days 6-8 from ovulation) and premenstrual (days 11-13 from ovulation). Initiation of testing was counterbalanced across the five phases. At the initiation of each session, each participant was requested to complete the state form of the State Trait Anxiety Inventory (STAI) [14]. During the first and last session, they were also instructed to fill out the trait form. Fifteen minutes following the participants' arrival to the laboratory, 10 ml of venous blood were collected from the antecubital vein in 5 ml vacutainer tubes containing 7.2 mg of EDTA. Blood samples were immediately centrifuged at 1,000 g for 10 minutes at 4°C to obtain the plasma fraction. Aiiquots of plasma were then taken from each sample, pipetted into culture tubes and stored at - 2 0 ° to -10°C until assayed for fl-endorphin by radioimmunoassay (RIA) using a commercially supplied kit (Immuno Nuclear Corporation No. 4600). Due to technical limitations, RIAs were completed only for Group 1. In brief, the RIA was performed in duplicate using the commercial protocol provided. Extraction of the samples was accomplished by first removing/3-LPH from the plasma with Sepharose anti-•-LPH. Next, the stripped plasma was extracted on ODS-silica columns with the eluent, methanol. The samples were then air-dried and reconstituted subsequently in BSA-borate buffer. Rabbit anti-Ct-endorphin serum was then added to the extracted plasma samples and then incubated for 24 hours. Next, labeled z2'~I-~-endorphin was added and another 24 hour incubation was instituted. Finally, goat anti-rabbit precipitating complex was added to precipitate the bound/3-endorphin antibody complex. Samples were then centrifuged after 10 hours incubation, the supernatant discarded, and the pellets counted via a

Beckman Gamma Scintillation Counter. Fhe counts were later entered into a computerized program designed fl)r Iogit analysis in order to determine the amount of/~-endorphin in the samples. This assay was sensitive to the 0.3-0.6 picogram range. While the assay had a 50~ cross reactivity with fl-LPH, this problem was circumvented by employing the J3-LPH extraction which removed this hormone from the plasma. The two methods of assessing pain thresholds were electric shock and a cold pressor task; presentation of each was counterbalanced across subjects. The electric shock paradigm consisted of application of concentric circular electrodes [17] to the inside of the participant's nondominant wrist. The electrode sponges were soaked with a saline solution and the participant's wrist was rubbed with an abrasive electrode paste. The amount of saline solution was adjusted until the current from a 1.5 volt battery was 50-+microamps. Shock was generated from a Tursky constant voltage shock source. Following application of the electrodes, a series of increasingly intense brief shocks (0-20 mA) was administered. The participant was instructed to tell the experimenter when she first detected the shock (pain detection) and when she experienced the shock as very unpleasant (pain threshold) at which point the procedure was terminated. To insure reliability, the scaling method was repeated twice and scores for both pain detection and pain thresholds were averaged across the three trials. The cold pressor task consisted of instructing the subject to immerse her dominant hand in a bucket of ice water (0°C) for as long as she could tolerate with a maximum of seven minutes. RESULTS In order to assess for the presence of an order effect resulting from the counterbalanced initiation of testing across the five sessions, data obtained from the dependent variables of pain detection, pain threshold, cold pressor and the state form of the STAI were separately analyzed using a 2 (group) × 5 (session) repeated measures analysis of variance. A similar 5 (session) analysis was completed with the plasma/~-endorphin data. Finally, a 2 (group) × 2 (Session 1 vs. Session 5) repeated measures analysis of variance was executed using the data obtained from the trait form of the STAI. These analyses revealed no significant differences resulting from the order of initiation of testing. A second group of repeated measures analyses of variance were executed to compare normally menstruating women with oral contraceptive users across the five menstrual phases. In addition to the 2 (group) × 5 (menstrual phase) analyses completed on the dependent variable listed above, a 5 (menstrual phase) analysis was performed with the plasma /3-endorphin data. None of the analyses achieved significance (see Table 1). Because of this, no simple effects tests comparing individual means were performed. It was observed that during the ovulatory phase, participants not only had a higher, though nonsignificant, mean level of circulating/3-endorphin but also a markedly higher variance during this period. An Fmax test was computed which revealed that the variances of fl-endorphin significantly differed across the menstrual cycle, Fro,x(5,8)= 28.48, p <0.01, with greatest variance present during the ovulatory phase and least during the luteal phase. It was also noted that the variance of Group 1 were markedly higher than those of Group 2 across the cold pressor task. Examination of individual data revealed that one nor-

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ENDORPHINS AND PAIN THRESHOLDS IN WOMEN TABLE 1 MEAN PERFORMANCEON MEASURESOF PAIN THRESHOLDS, ANXIETY AND PLASMA/3-ENDORPHIN Menstrual Phase Pain Detection (mA) Group 1 Mean S.D. Group 2 Mean S.D. Pain Thresholds (mA) Group 1 Mean S.D. Group 2 Mean S.D. Cold Pressor (sec) Group 1 Mean S.D. Group 2 Mean S.D. State Anxiety Group 1 Mean S.D. Group 2 Mean S.D. /3-Endorphin (pg/ml) Group 1 Mean S.D.

Menstrual

Follicular

0.80 0.59

0.63 0.29

0.76 0.50

0.69 0.36

0.67 0.39

0.50 0.24

0.49 0.24

0.62 0.25

0.75 0.41

0.75 0.50

5.95 5.66

5.71 4.20

5.14 4.13

6.74 4.47

6.10 3.87

4.43 1.67

4.23 2.22

4.81 1.88

5.73 2.84

5.66 3.75

72.50 131.63

82.00 137.62

68.00 133.14

64.06 133.85

71.75 131.99

41.00 31.21

40.07 35.28

34.93 24.00

39.07 36.43

32.29 22.97

36.56 10.28

34.22 7.60

34.89 6.37

42.33 14.54

38.50 9.26

37.86 11.02

36.71 6.40

34.29 6.02

36.57 12.46

35.29 3.73

4.94 7.00

8.83 13.31

40.64 96.20

7.50 5.70

9.98 8.18

mal cycling participant, a marathon swimmer, consistently scored the maximum seven minutes on this task. With this subject excluded, a 2 (group) x 5 (menstrual phase) repeated measures analysis of variance revealed no significant differences between groups across this variable and also, the variances between the two groups did not vary significantly. To further examine the possible relationships between plasma fl-endorphin levels and the behavioral measures for Group l, Pearson product-moment correlations were computed using/3-endorphin levels as one variable and pain detection, pain threshold, cold pressor, or state anxiety as the other variable. Of interest, fl-endorphin levels were significantly positively correlated with state anxiety, r(7)=.798, p<0.01, during the menstrual phase. All other correlations were nonsignificant. DISCUSSION The markedly greater degree of variance in peripheral /3-endorphin levels found during the ovulatory phase in normally menstruating women as compared to levels of this hormone during other phases of the menstrual cycle is of

Ovulatory

Luteal

Premenstrual

interest. Since ovulation is a relatively discrete phenomenon in time [9], one might conjecture that the extremely high elevations of fl-endorphin obtained from several of the participants may have been due to the fact that they were tested exactly at ovulation. However, since this study did not employ measures of gonadotropins or ovarian steroids but relied on basal body temperature to predict occurrence of ovulation, it is impossible to determine the exact nature of the cause of the scattered findings of extreme elevations during the ovulatory phase. Further work designed to assess the relationship of peripheral fl-endorphin levels and the HPG axis is needed. Moreover, this finding does suggest that the menstrual cycle should be considered when studying the endorphin system in women. Statistical examination of the obtained variances across the variable of plasma fl-endorphin revealed that during the ovulatory phase, normal cycling women exhibited the greatest variance as compared to the luteal phase when the least variance occurred. The variances of the other phases did not significantly differ from the luteal phase. On this basis, it is recommended that great care be

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VEI'I-H E l A L

employed when studying normally menstruating w o m e n at midcycle, due to the possibility of extreme elevations of plasma levels of this peptide occurring at this time. Another report [20] has found significant variation in /3-endorphin levels in the hypophyseal portal blood of nonhuman primates. Of interest, levels were most elevated during those phases surrounding ovulation (follicular and luteal). To the authors' knowledge, the degree of correlation b e t w e e n central and peripheral levels of this peptide is presently unknown. Thus, as might be predicted, the present study examining peripheral variation did not obtain results which exactly mirrored the pattern of central changes of this peptide. Further work studying/3-endorphin levels in female human C S F , is called for in order to delineate if this variance in findings resulted from the different species e m p l o y e d and/or the use of central as c o m p a r e d to peripheral measures. The counterbalanced initiation of testing did not result in an order effect across any of the dependent variables. This is surprising insofar as the experimental paradigm had a n u m b e r of seemingly aversive elements, including venipunctures, electric shock and the cold pressor task. The absence of an order effect across such potentially anxiety sensitive measures as the STAI and plasma /3-endorphin levels may be interpreted in a number of ways. It is possible that the participants did not habituate to the stressful elements of the study, that they did not perceive the paradigm as anxiety provoking, or that the dependent variables employed were inadequate measures of this emotional dimension. Furthermore, the results of this study are in direct opposition to other reports [5, 7, 12, 16] indicating reliable chan~es in the ~ensory modality of oain response occurring in normally menstruating w o m e n . The reasons for the lack of

replication are unclear but the most likely possibility is the e m p l o y m e n t of a paradigm which differs markedly from the other cited studies. Brief electric shock and the cold pressor task were used in this study for the reasons that no earlier reports of menstrually-related variability in pain response to these types of stimuli is available and since they are widely e m p l o y e d pain induction paradigms. The findings of the present study suggest that cyclic variation in pain thresholds is highly dependent on the method e m p l o y e d [2,15] and is not a robust p h e n o m e n o n occurring across all types of painful stimuli. The significant positive correlation of plasma/3-endorphin and state anxiety during the menstrual phase but not other phases of the menstrual cycle in normally menstruating w o m e n is suggestive. Menstruation may be considered the phase characterized by relatively depressed levels of gonadotropins and ovarian steroids [9]; this unique hormonal e n v i r o n m e n t may interact with/3-endorphin's relationship to experienced anxiety. During the menstrual phase, as /3-endorphin levels increase so does negative affect: if this is a causal relationship is unclear at this time. In summary, peripheral/3-endorphin levels are relatively stable across the menstrual cycle with the exception of the ovulatory phase which is characterized by scattered, marked elevations of this peptide. Pain thresholds, as measured by brief electric shock and cold pressor task, do not fluctuate in a consistent pattern across the menstrual cycle nor is response to these painful stimuli positively related to circulating/3-endorphin levels. H o w e v e r , reported anxiety appears to be related to levels of this hormone during the period of menstruation. The results of this study suggest that the menstrual cycle should be considered when examining the functioning of the endorphin system in normally menstruating women.

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