0021-972x/92/7503-0924%03.M)/0 Journal of Clinical Endocrinology and Metabolism Copyright 0 1992 by The Endocrine Society
Vol. 15, No. 3 Printed in U.S.A.
Plasma Neurotransmitter Phases of the Ovulatory ILANA BLUM, LIORA LOUISE WEISSGLAS, AND YAFFA VERED
Profile Cycle
NESSIEL, AMNON DAVID, URI GABBAY, JACQUELINE
during
Different
ERAN GRAFF, ANNA HARSAT, SULKES, YAIR YERUSHALMY,
Institute of Endocrinology and Metabolism, Beilinson Medical Center (Z.B.), Petah Tiqva; the Suckler School of Medicine, Tel Aviv University (Z.B., A.D., E.G.), Ramat Aviv; the Clinical Chemistry Department, Tel Aviv Sourasky Medical Center, Zchilov Hospital (L.N., E.G., A.H., Y. V.), the Zamenhoff Kupat Holim Outpatient Clinic (A.D., L. W., Y. Y.), Tel Aviv; and the Epidemiology Unit, Beilinson Medical Center (U.G., J.S.), Petah Tiqva, Israel ABSTRACT The influence of the different phases of the menstrual cycle on platelet-poor plasma norepinephrine (NE) and serotonin (5HT) was examined in 17 normal volunteers. The examinations were performed consecutively during 3 phases of the ovulatory cycle: 1) follicular phase, 2) ovulation, and 3) luteal phase. This investigation was initiated after a preliminary study in 51 volunteers showed wide and consistent variations of plasma NE and 5HT during the different phases of the cycle. Since in this first group the determinations had not been performed consecutively in the same subjects, and the changes observed in the different phases of the cycle could reflect interpersonal variations, the determinations were performed consecutively in a second group, concomitantly with serum
estradiol (EJ and LH measurements. The results showed a decrease in plasma 5HT from the follicular phase [144.3 -+ 69.3 nmol/L (SD)] to ovulation (55.7 + 41.4; P < 0.001) and a subsequent increase in the luteal phase (141.3 f 96.4; P < 0.01). The nadir in plasma 5HT showed an inverse correlation with serum LH (r = -0.07). Plasma NE increased from the follicular phase (1226.5 + 475.1 pmol/L) to ovulation (1694.0 + 564.4; P = 0.027) and reached a maximum in the luteal phase (2335.0 f 728.2; P = 0.0034). This rise correlated positively with serum E,. In conclusion, plasma 5HT and NE vary with the different phases of the menstrual cycle. Plasma NE rises during ovulation and seems to to correlate positively with serum E, levels. Plasma 5HT reaches a nadir during ovulation and correlates inversely with serum LH. (J Clin Endocrinol Metub 75: 924-929, 1992)
M
of 51 normal volunteers, aged 32.9 + 1.6 yr, with normal body weights (body mass index, 25.5 + 4.09 kg/m2). In view of the results obtained in this group, a second group (group B), consisting of 17 volunteers of comparable age (32.9 + 5.1 yr) and body weight (body mass index, 25.6 + 3.0 kg/m*) was selected. In this group plasma neurotransmitters were examined consecutively in the same woman during the different phases of the ovulatory cycle concomitantly with serum estradiol (E2) and LH. None of the women had a history of any organic or mental diseases. They had regular menstrual cycles. They had not been pregnant within the last year and had not taken any medication for 3 months before the study. All of them had similar levels of physical activity. Each woman gave a detailed medical history and had a thorough physical examination. The different phases of the menstrual cycle were noted as follows: 1) days l-5, menstruation; 2) days 6-l 1, follicular phase; 3) days 12-16, ovulation; and 4) days 17-22, luteal phase. In group A plasma was drawn for neurotransmitter determinations on only one occasion during the cycle. The phase of the cycle was noted, and the results were averaged for each phase. In group B blood was collected during the follicular phase and, starting from day 10, every second day until day 22; the results were averaged for each phase. The different phases of the cycle were determined by measuring basal body temperature, Ez, LH, and progesterone. Plasma was collected during the same season at 0800 h after a 14-h fast in order to avoid possible seasonal, circadian, or meal-induced variations. No caffeine-containing beverages were consumed, and no cigarettes were smoked for 14 h before blood collection. Blood was collected from supine subjects 0 and 30 min after the venipuncture. The collection was performed through an indwelling needle in refrigerated tubes containing Na-EDTA and Na-metabisulfite. To obtain PPP, the tubes were centrifuged at 4 C immediately after the collection at 1500 X g for 20 min (14). The supernatant was stored at -70 C until assayed.
ONOAMINE neurotransmitters [norepinephrine (NE) and serotonin (5HT)] have been shown to influence mood (1, 2), various metabolic processes(3), the secretion of various hormones (4-6), and ovulation (7, 8). In addition, they have been implicated in the etiopathogenesis of pathological processes,such as psychiatric disturbances (1, 2), increased blood pressure (9, lo), myocardial ischemia (ll), etc. Plasma levels of NE and 5HT may be influenced by various factors, such as diet (12), exercise (13), drugs (1416), estrogens(17-19), etc. Several reports have shown that plasma NE varies predictably during the different phasesof the ovulatory cycle (20-22). However, no such variations have been demonstrated so far for plasma 5HT. In view of the important role of NE and 5HT in the ovulatory process and the fact that platelet-poor plasma (PPP) neurotransmitter levels seem to reflect processesoccurring in the brain (12, 14, 23), it is the purpose of this report to show the variations in NE and, for the first time, in 5HT during the menstrual cycle in the PPP of normal young women. Subjects and Methods The influence of the different phases NE and 5HT was studied in two groups Received Address docrinology,
of the menstrual cycle on PPP of patients. Group A consisted
September 5, 1991. requests for reprints to: I. Blum, M.D., Beilinson Medical Center, Petah Tiqva
Department of En49 100, Israel.
924
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PLASMA Analytical
NEUROTRANSMITTERS
THE
OVULATORY
CYCLE
925
methods
PPP concentrations of 5HT (24) and NE (BioAnalytical Systems application note 14) were determined by a high performance liquid chromatography with electrochemical detection method. The high performance liquid chromatography system consisted of an LDC-Milton Roy high pressure pump, a Rheodyne injector, a reverse phase chromatographic column (BioAnalytical Systems)-octadecyl silica column (4 x 250 mm; 5 pm), and an electrochemical detector (LC-4, BioAnalytical Systems) equipped with TL-5 glassy carbon electrode. The applied potential at the working electrode was +0.64 V. The mobile phase consisted of a monochloroacetate buffer containing 2 rnM EDTA. For NE determinations, an ion pair agent (sodium octyl sulfate; 50-100 mg/L) was added. For 5HT determinations, methanol was added at a final concentration of 5%. Serum hormones were determined by RIA, using commercially available kits. Ez was measured by the double antibody technique of RSL (ICN Biochemicals, Inc., CA). The method was modified for increased sensitivity by increasing the length of incubation to 18 h at room temperature. LH was measured by RIA using a double antibody technique, with magnetic separation using a kit of Amerlex-M (Amersham, Aylesbury, Buckinghamshire, United Kingdom).
Statistical
AND
0 M Ia F. P.
150 n=lZ
5 ; 100
H
0
ml
L.P.
ml9
n=12
analysis
Statistical analysis was performed using the one-way analysis of variance between PPP 5HT and PPP NE concentrations during the different phases of the ovulatory cycle. To determine the significance of the differences in the mean PPP 5HT and PPP NE levels in each phase of the cycle, the multiple comparison analysis was employed, using the general linear model procedure of the statistical analysis software (SAS Institute, Cary, NC). The correlation between the neurotransmitter changes and the changes in E2 and LH was determined using the Pearson correlation coefficient as a measure of direction and strength of association. A test consistent with P < 0.05 was considered significant.
Results Group A
Plasma 5HT was highest during the menstrual period (136.25 f 112.3 nmol/L). It decreasedto 66.3 + 37.8 nmol/ L (SD; P < 0.03) during the follicular phase, reached a nadir during ovulation (29.9 + 23.1 nmol/L; P < 0.27), and started to increaseagain during the luteal phase (69.6 + 70.9 nmol/ L; P < 0.14; Fig. 1). Plasma NE during the menstrual phase was 889.52 f 407.15 pmol/L. It decreased during the follicular phase to 474.08 f 314.8 pmol/L (P = 0.0787), rose during ovulation to 876.4 + 629.34 pmol/L (P = 0.09), and peaked during the luteal phase at 1053.25 + 480.5 pmol/L (P < 0.0099 compared to the follicular phase value; Fig. 2). Unless stated otherwise, the P values cited in the above paragraphs represent comparisons to the previous cycle stage. In addition, the multiple comparisons between all stagesare shown in Figs. 1 and 2. Since the results in group A could also have reflected interpersonal variations, a secondgroup of 17 women (group B) was selected. In this group the determinations were performed consecutively in the samewoman during the different phases of the ovulatory cycle, as previously described. The results in this group represent the mean + SD of all determinations performed during the same phase of the ovulatory cycle. It was remarkable that the plasma neurotransmitter levels showed very little variation when meas-
0
L
El
FIG. 1. Mean & SD PPP 5HT levels during the different ovulatory cycle in group A subjects. M, Menstruation; phase; 0, ovulation; L.P., luteal phase.
m
phases of the F.P., follicular
ured in the same patient on the same day or on different days of the samephase of the cycle (results not shown). The values (mean + SD) for PPP-5 HT during the different phases of the cycle as well as the results of the multiple comparison analysis are shown in Fig. 3. From this figure it is seenthat essentially similar results were obtained asin the first group. However, in this group the differences between the phasesof the cycle were statistically significant. When a one-way analysis of variance between PPP 5HT level and phase of the ovulatory cycle was performed, a statistically significant difference between the phases of the ovulatory cycle was achieved (P < 0.001). The phase variation of the ovulatory cycle explains 25% of the variation in plasma 5HT (r’ = 0.25). The difference between the mean values of plasma 5HT in the follicular phase and during ovulation was statistically significant (P < O.OOl), similar to that between ovulation and the luteal phase (P < 0.01). However, no significance was observed between the follicular and luteal phases (Fig. 3). The mean + SD and the results of the multiple comparison analysis of plasma NE during the different phases of the cycle are shown in Fig. 4. The one-way analysis of variance between plasma NE and the phasesof the cycle showed an overall statistically significant difference (P < 0.0001). The changes induced by the different phases of the ovulatory cycle explain 38% of the variation in plasma NE (r’ = 0.38). The increase in plasma NE from the follicular phase to
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926
BLUM
ET AL.
El M
lzl I3
tzl FsP. I3 0
F.R 0
•II L.P.
ml L.P.
n=9
n=17
n=8
m
JCE & M. 1992 VolX.No3
Ei
k17
FIG. 2. Mean + SD PPP NE levels during the different ovulatory cycle in group A subjects. M, Menstruation; phase; 0, ovulation; L.P., luteal phase.
phases of the F.P., follicular
ovulation was significant (P < 0.05), as was the increase from ovulation to the luteal phase (P < 0.01; Fig. 4). Figure 5 shows the mean change over baseline in plasma 5HT, plasma NE, serum EZ,and serum LH during the different phasesof the cycle. From this figure it is seen that the nadir in PPP 5HT during ovulation coincides with the maximal values of serum LH. The overall negative correlation between PPP 5HT and serum LH (-0.07) may indicate that while PPP 5HT increases, serum LH decreases.An overall positive correlation was observed between plasma NE (0.22) and serum Ez. Discussion
The results obtained in group A show that plasma neurotransmitters varied during the menstrual cycle. However, since these values could also have been the result of interpersonal variations, the samemeasurementswere performed consecutively in the samewoman during the different phases of the cycle in 17 subjects (group B) and correlated with hormonal variations. In both groups the neurotransmitter levels varied consistently during the different phasesof the menstrual cycle. Results similar to ours were obtained by Zuspan and Zuspan (20) and Goldstein et al. (22), who found an increase in plasma NE, 2 days before ovulation and a continued increaseafter ovulation, so that the average luteal phase plasma NE level was significantly higher than the
3. Mean + SD PPP 5HT ovulatory cycle in the group ovulation; L.P., luteal phase.
FIG.
levels during B subjects.
the different phases of the F.P., Follicular phase; 0,
follicular phase NE concentration (22). The variations in plasmaNE levels may be attributed to the effects of estrogens on catecholamine degradation (18). It has been shown that estrogens impair NE presynaptic reuptake, compete with catecholaminesfor the degradative enzyme catechol-o-methyltransferase (18), and enhance the rate of monoamineoxidase degradation (25), thereby reducing its activity. Thus, the increase in plasma NE during ovulation and the luteal phase of the cycle observed by us may be explained by an increase in serum estrogen. Another explanation might be linked to the possibility of the plasma levels reflecting processesoccurring in the hypothalamus, such that the above described variations might reflect the hypothalamic NE surge preceding ovulation (7, 26, 27). The variations in PPP 5HT during the menstrual cycle have not been reported previously. Conflicting evidence exists for the role of estrogens in regulation of plasma 5HT
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PLASMA
NEUROTRANSMITTERS
AND
THE
OVULATORY
CYCLE
927
tzl F.P. f
q O ml L.P.
-------LH
:
ml7
i I I
EliI
-NF
iP. FIG. 4. Mean f SD PPP NE levels during ovulatory cycle in the group B subjects. ovulation; L.P., luteal phase.
the different phases of the F.P., Foliicular phase; 0,
levels (28, 29). Theoretically, estrogens should decrease central 5HT by three pathways. 1) Activation of tryptophan 2,3dioxygenase in the liver and the peripheral kynurenine pathway (28) may decrease Trp levels available for 5HT synthesis. B) The second pathway is linked to the previous one; kynurenine may compete with Trp for passage through the bloodbrain barrier (30) and decrease the substrate available for central 5HT synthesis. 3) Estrogens produce a relative absence of pyridoxine, which is an essential cofactor in the activity of L-aromatic acid decarboxylase, one of the enzymes crucial for 5HT synthesis (31, 32). Since the action of PPP 5HT has been shown to parallel phenomena occurring in the brain (12, 14), a decrease in central 5HT might be expected to be accompanied by low PPP 5HT levels. On the other hand, estrogens have also been shown to produce opposite effects by increasing the rate of monoamineoxidase degradation (25) and displacing Trp from its binding sites to plasma albumin (19). In this connection, PPP 5HT has been shown to decrease after ovariectomy (29) and menopause (33). In our two groups of patients we observed large differences in PPP 5HT levels between the ovulatory and luteal phases,
i,
LI.P.
FIG. 5. The correlation between PPP 5HT and NE variations and E, and LH during the different phases of the ovulatory cycle. The results are expressed as the mean change over baseline. F.P., Follicular phase; 0, ovulation; L.P., luteal phase.
when similar estrogen levels were observed. No consensus has been reached in the literature for the role of 5HT in GnRH and gonadotropin secretion (8, 34). However, the most convincing evidence has been adduced for an inhibitory role of 5HT on GnRH and gonadotropin secretion (35,36). Ovulation occurs only when this inhibitory influence disappears. This latter theory might be in concordance with the significant drop in the PPP 5HT level during ovulation and the significant inverse correlation between PPP 5HT and serum LH. However, a peripheral effect of LH on PPP 5HT cannot be ruled out by the present study. High plasma NE levels during menstruation have already been reported by Feichtinger et al. (21). In our group A patients similar high levels of plasma NE and 5HT were observed. It is possible that the high levels of PPP 5HT and NE during this period are explained by the stress produced by menstrual pains. The increased stress levels might produce a secondary increase in adrenal catecholamine output as a result of the increase in glucocorticoid levels (37). However, this mechanism does not explain the increase in PPP 5HT due to glucocorticoids decreasing 5HT levels by diverting its
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928
BLUM
precursor to the kynurenine pathway (38). Another more plausible explanation might be linked to the peripheral release of 5HT and NE from the platelets during menstruation. In conclusion, plasma neurotransmitter levels have been shown to vary predictably during the menstrual cycle. Plasma NE levels are lowest during the follicular phase and highest during the luteal phase and menstruation; plasma 5HT levels are highest during menstruation and lowest during ovulation. These findings should be taken into consideration in the design and analysis of studies that use PPP 5HT and NE as indicators of serotonergic and adrenergic activities in different diseases. At this stage there is no clear-cut evidence linking the changes in plasma neurotransmitter profile, observed by us, to hormonal, neuronal, or central neurotransmitter changes. However, several lines of evidence indicate that PPP 5HT and plasma NE reflect central processes. Results from Artigas’s laboratory showed the existence of a freely circulating pool of 5HT in the plasma different from that in the platelets (39,40). This pool reacts differently to chronic drug treatment than does the platelet pool. Thus, chronic treatment with lithium salts in bipolar patients increased PPP 5HT levels, while leaving platelet 5HT concentrations unchanged (14). Results obtained in our laboratory showed that changes in meal composition producing changes in the availability of tryptophan (the precursor of 5HT synthesis) for transport through the blood-brain barrier induced changes in PPP 5HT similar to those reported in the brain (12,41). Taken together these results indicate that PPP 5HT may be a useful peripheral model to study brain processes mediated by 5HT. Plasma NE has already been shown to reflect neuronally occurring processes (23). These facts concur with the results reported by Goldstein et al. (22), who showed an increase in plasma NE before ovulation, in suggesting that changes in the plasma neurotransmitter profile reflect hypothalamic processes linked to ovulation. Thus, the increase in EZ levels before ovulation that produces an increase in hypothalamic NE, which mediates the GnRH surge (7), seems to be reflected in the plasma levels. However, much more work is needed to explore these questions. Our findings might serve to elucidate different pathological states linked to the menstrual cycle. In addition, it is possible that in the future these findings might help women suffering from mood disorders linked to the menstrual cycle or women experiencing different anovulatory states. References 1. Meltzer HY, Lowy MT. 1987 The serotonin hypothesis of depression. In: Meltzer HY, ed. Psychopharmacology: third generation of progress. New York: Raven Press; pp 513-26. 2. Schildkraut JJ. 1965 The catecholamine hypothesis of affective disorders: a review of supportive evidence. Am J Psychiatry. 122:509-22. 3. Campese VM, De Quattro V. 1989 Functional components of the sympathetic nervous system: regulation of organ systems. In: De Groot LJ, ed. Endocrinology, 2nd ed, Philadelphia: Saunders; pp 1738-56. 4. Gallo RV. 1980 Neuroendocrine regulation of pulsatile luteinizing hormone in the rat. Neuroendocrinology. 30:122-31. 5. Kreiger HB, Krieger DT. 1970 Chemical stimulation of the brain:
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effect on adrenal corticoid release. Am J Physiol. 218:16-32. 6. Rees LH, Butler PWP, Gosling C, Besser GM. 1970 Adrenergic blockade and the corticosteroid and growth hormone responses to methylamphetamine. Nature. 288:565-6. 7. Barraclough CA, Wise PM. 1982 The role of catecholamines in the regulation of pituitary Iuteinizing hormone and follicle-stimulating hormone secretion, Endocr Rev. 3:91-119. 8. Hery M, Laplant E, Kordon C. 1976 Participation of serotonin in phasic release of LH. I. Evidence from pharmacological experiments, Endocrinology. 99:496-503. 9. Van Nutten JM, Janssen WJ, Janssen PAJ. 1990 Altered response to 5-hydroxytriptamine in hypertension and other cardiovascular disorders. In: Saxena PR, Wallis DI, Wouters W, Bevan P, eds. Cardiovascular pharmacology of 5-hydroxytriptamine prospective therapeutic evaluation. Dordrecht, Boston, London: Kluwer Academic; pp 303-7. 10. De Quattro V, Miura Y. 1973 Neurogenic factors in hypertension: mechanism or mvth? Am I Med. 55:362-78. 11. Hillis LD, Lange RA. 1991 Serotonin and acute ischemic heart disease. N Engl J Med. 324:688-9. 12. Blum I, Vered Y, Graff E, Harsat A, Grosskopf Y, Raz 0. 1992 The influence of meal composition on plasma serotonin and norepinephrine concentrations. Metabolism. 41:137-40. 13. Dimsdale JE, Moss J. 1980 Plasma catecholamines in stress and exercise. JAMA. 243:340-2. 14. Artigas F, Sarrias MJ, Martinez E, et al. 1989 Increased plasma free serotonin but unchanged platelet serotonin in bipolar patients treated chronically with lithium. Psychopharmacology. 99:328-32. 15. De Quattro V, Miura Y, Campese V, Brunjes D. 1975 Catecholamine biosynthesis in man: effects of hypertension and reserpine. In: Millier I’, Safar M, eds. Recent advances in hypertension, Paris: Laboratories Boehringer Ingelheim; vol 1:13-26. 16. Esler M, Julius S, Zweifler A, et al. 1977 Mild high-renin essential hypertension: a neurogenic human hypertension? N Engl J Med. 296:405-l 1. 17. Blum M, Zacharovich D, Gelernter I, Blum I. 1988 Influence of oral contraceptive treatment on blood pressure and 24-hour urinary catecholamine excretion in smoking as compared with non-smoking women. Adv Contraception. 4:143-9. 18. Brener H, Koster G, Schneider ST, Ladowsky W. 1978 Interactions between estrogens and neurotransmitters. Effects of estrogens on the enzymaticmethylation of noradrenaline in the brain. In: Scott DE, Roslowski GE, Weindl A, eds. Brain endocrine interactions. Base]: Karger; vol 3:274-85. 19. Burton RM, Westphal U. 1972 Steroid hormone binding proteins in blood plasma. Metabolism. 25:253-76. 20. Zuspan FP, Zuspan KJ. 1973 Ovulatory plasma amine (epinephrine and norepinephrine) surge in the woman. Am J Obstet Gynecol. 117:654-l. 21. Feichtinger W, Keukter P, Salzer H, Euller A, Korn A, Friedrich F. 1980 Katecholaminausscheidung im Harn bei Frauen mit normalen Menstruation-Zyklus. Wien Klin Wochenschr. 92:365-8. 22. Goldstein DS, Levinson P, Keiser HR. 1983 Plasma and urinary catecholamines during the human ovulatory cycle. Am J Obstet Gynecol. 146:824-9. 23. Floras JS, Legault L, Moralli GA, Hara K, Blendis LM. 1991 Increased sympathetic outflow in cirrhosis and ascites: direct evidence from intraneural recordings. Ann Intern Med. 114:373-80. 24. Tagari PC, Boullin DJ, Davies CL. 1984 Simplified determination of serotonin in plasma by liquid chromatography. Clin Chem. 30:131-5. 25. Klaiber EL, Kobayashi Y, Braverman DM, Hall F. 1971 Plasma monoamineoxidase activity in regularly menstruating women and in amenorrheic women receiving cyclic treatment with estrogens and a progestin. J Clin Endocrinol Metab. 33:630-B. 26. Weiner RI, Ganong WI. 1978 Role of brain monoamines and histamine in regulation of anterior pituitary secretion. Physiol Rev. 58:905-76. 27. Rabii J, Ehlers I’, Clifton D, Sawyer CH. 1980 Effects of intraventricular infusion of 6-hydroxydopamine (6-OHPA) on pituitary LH release and ovulation in the rabbit. Neuroendocrinology. 30:362-8. 28. Hrboticky N, Leiter LA, Anderson GH. 1989 Menstrual cycle
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Guicheney P, Marcel D. 1984 Decreased platelet serotonin content in ovariectomized female rats. Endocrinology. 114:2412-4. 30. Moller SE. 1981 Pharmacokinetics of tryptophan, renal handling of kynurenine and the effect of nocotinamide on its appearance in plasma and urine following L-tryptophan loading of healthy subjects. Eur J Clin Pharmacol. 21:137-42. 31. Lekleni JE. 1971 Quantitative aspects of tryptophan metabolism in humans and other species: a review. Am J Clin Nutr. 24:659-72. 32. Wolf H. 1974 Vitamin B6 and the kynurenine pathway. Stand J Clin Lab Invest. 33(Suppl):63-76. 33. Gonzales GF. 1980 Blood levels of 5-hydroxytryptamine in human beings under several physiological situations. Life Sci. 27:647-50. 34. Delitala G. 1989 Clinical neuropharmacology in the management of disorders of the pituitary and hypothalamus. In: De Groot LJ, ed. Endocrinology, 2nd ed. Philadelphia: Saunders; pp 454-73. 35. McCann SM. 1982 Physiology and pharmacology of LHRH and
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37. De Quattro V, Myers M, Campese VM. 1989 Anatomy and biochemistry of the sympathetic nervous system. In: De Groot LJ, ed. Endocrinology, 2nd ed. Philadelphia: Saunders; pp 1717-37. 38. Badaway AAB. 1977 The function and regulation of tryptophan pyrrolase.
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E, Gelpi E. 1985 Serotonin in body fluids: characterization of human plasma and cerebrospinal fluid pools by means of a new HPLC method. Life Sci. 37:441-7. Ortiz J, Artigas F, Gelpi E. 1988 Serotonergic status in human blood. Life Sci. 43:983-90. Wurtman RJ. 1988 Effect of their nutrient precursors on the synthesis and release of serotonin, the catecholamines, and acetylcholine: implications for behavioral disorders. Clin Neuropharmacol. 1 l:s8-~32.
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Vol. 15, No. 3 Printed in U.S.A.
Plasma Neurotransmitter Phases of the Ovulatory ILANA BLUM, LIORA LOUISE WEISSGLAS, AND YAFFA VERED
Profile Cycle
NESSIEL, AMNON DAVID, URI GABBAY, JACQUELINE
during
Different
ERAN GRAFF, ANNA HARSAT, SULKES, YAIR YERUSHALMY,
Institute of Endocrinology and Metabolism, Beilinson Medical Center (Z.B.), Petah Tiqva; the Suckler School of Medicine, Tel Aviv University (Z.B., A.D., E.G.), Ramat Aviv; the Clinical Chemistry Department, Tel Aviv Sourasky Medical Center, Zchilov Hospital (L.N., E.G., A.H., Y. V.), the Zamenhoff Kupat Holim Outpatient Clinic (A.D., L. W., Y. Y.), Tel Aviv; and the Epidemiology Unit, Beilinson Medical Center (U.G., J.S.), Petah Tiqva, Israel ABSTRACT The influence of the different phases of the menstrual cycle on platelet-poor plasma norepinephrine (NE) and serotonin (5HT) was examined in 17 normal volunteers. The examinations were performed consecutively during 3 phases of the ovulatory cycle: 1) follicular phase, 2) ovulation, and 3) luteal phase. This investigation was initiated after a preliminary study in 51 volunteers showed wide and consistent variations of plasma NE and 5HT during the different phases of the cycle. Since in this first group the determinations had not been performed consecutively in the same subjects, and the changes observed in the different phases of the cycle could reflect interpersonal variations, the determinations were performed consecutively in a second group, concomitantly with serum
estradiol (EJ and LH measurements. The results showed a decrease in plasma 5HT from the follicular phase [144.3 -+ 69.3 nmol/L (SD)] to ovulation (55.7 + 41.4; P < 0.001) and a subsequent increase in the luteal phase (141.3 f 96.4; P < 0.01). The nadir in plasma 5HT showed an inverse correlation with serum LH (r = -0.07). Plasma NE increased from the follicular phase (1226.5 + 475.1 pmol/L) to ovulation (1694.0 + 564.4; P = 0.027) and reached a maximum in the luteal phase (2335.0 f 728.2; P = 0.0034). This rise correlated positively with serum E,. In conclusion, plasma 5HT and NE vary with the different phases of the menstrual cycle. Plasma NE rises during ovulation and seems to to correlate positively with serum E, levels. Plasma 5HT reaches a nadir during ovulation and correlates inversely with serum LH. (J Clin Endocrinol Metub 75: 924-929, 1992)
M
of 51 normal volunteers, aged 32.9 + 1.6 yr, with normal body weights (body mass index, 25.5 + 4.09 kg/m2). In view of the results obtained in this group, a second group (group B), consisting of 17 volunteers of comparable age (32.9 + 5.1 yr) and body weight (body mass index, 25.6 + 3.0 kg/m*) was selected. In this group plasma neurotransmitters were examined consecutively in the same woman during the different phases of the ovulatory cycle concomitantly with serum estradiol (E2) and LH. None of the women had a history of any organic or mental diseases. They had regular menstrual cycles. They had not been pregnant within the last year and had not taken any medication for 3 months before the study. All of them had similar levels of physical activity. Each woman gave a detailed medical history and had a thorough physical examination. The different phases of the menstrual cycle were noted as follows: 1) days l-5, menstruation; 2) days 6-l 1, follicular phase; 3) days 12-16, ovulation; and 4) days 17-22, luteal phase. In group A plasma was drawn for neurotransmitter determinations on only one occasion during the cycle. The phase of the cycle was noted, and the results were averaged for each phase. In group B blood was collected during the follicular phase and, starting from day 10, every second day until day 22; the results were averaged for each phase. The different phases of the cycle were determined by measuring basal body temperature, Ez, LH, and progesterone. Plasma was collected during the same season at 0800 h after a 14-h fast in order to avoid possible seasonal, circadian, or meal-induced variations. No caffeine-containing beverages were consumed, and no cigarettes were smoked for 14 h before blood collection. Blood was collected from supine subjects 0 and 30 min after the venipuncture. The collection was performed through an indwelling needle in refrigerated tubes containing Na-EDTA and Na-metabisulfite. To obtain PPP, the tubes were centrifuged at 4 C immediately after the collection at 1500 X g for 20 min (14). The supernatant was stored at -70 C until assayed.
ONOAMINE neurotransmitters [norepinephrine (NE) and serotonin (5HT)] have been shown to influence mood (1, 2), various metabolic processes(3), the secretion of various hormones (4-6), and ovulation (7, 8). In addition, they have been implicated in the etiopathogenesis of pathological processes,such as psychiatric disturbances (1, 2), increased blood pressure (9, lo), myocardial ischemia (ll), etc. Plasma levels of NE and 5HT may be influenced by various factors, such as diet (12), exercise (13), drugs (1416), estrogens(17-19), etc. Several reports have shown that plasma NE varies predictably during the different phasesof the ovulatory cycle (20-22). However, no such variations have been demonstrated so far for plasma 5HT. In view of the important role of NE and 5HT in the ovulatory process and the fact that platelet-poor plasma (PPP) neurotransmitter levels seem to reflect processesoccurring in the brain (12, 14, 23), it is the purpose of this report to show the variations in NE and, for the first time, in 5HT during the menstrual cycle in the PPP of normal young women. Subjects and Methods The influence of the different phases NE and 5HT was studied in two groups Received Address docrinology,
of the menstrual cycle on PPP of patients. Group A consisted
September 5, 1991. requests for reprints to: I. Blum, M.D., Beilinson Medical Center, Petah Tiqva
Department of En49 100, Israel.
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PLASMA Analytical
NEUROTRANSMITTERS
THE
OVULATORY
CYCLE
925
methods
PPP concentrations of 5HT (24) and NE (BioAnalytical Systems application note 14) were determined by a high performance liquid chromatography with electrochemical detection method. The high performance liquid chromatography system consisted of an LDC-Milton Roy high pressure pump, a Rheodyne injector, a reverse phase chromatographic column (BioAnalytical Systems)-octadecyl silica column (4 x 250 mm; 5 pm), and an electrochemical detector (LC-4, BioAnalytical Systems) equipped with TL-5 glassy carbon electrode. The applied potential at the working electrode was +0.64 V. The mobile phase consisted of a monochloroacetate buffer containing 2 rnM EDTA. For NE determinations, an ion pair agent (sodium octyl sulfate; 50-100 mg/L) was added. For 5HT determinations, methanol was added at a final concentration of 5%. Serum hormones were determined by RIA, using commercially available kits. Ez was measured by the double antibody technique of RSL (ICN Biochemicals, Inc., CA). The method was modified for increased sensitivity by increasing the length of incubation to 18 h at room temperature. LH was measured by RIA using a double antibody technique, with magnetic separation using a kit of Amerlex-M (Amersham, Aylesbury, Buckinghamshire, United Kingdom).
Statistical
AND
0 M Ia F. P.
150 n=lZ
5 ; 100
H
0
ml
L.P.
ml9
n=12
analysis
Statistical analysis was performed using the one-way analysis of variance between PPP 5HT and PPP NE concentrations during the different phases of the ovulatory cycle. To determine the significance of the differences in the mean PPP 5HT and PPP NE levels in each phase of the cycle, the multiple comparison analysis was employed, using the general linear model procedure of the statistical analysis software (SAS Institute, Cary, NC). The correlation between the neurotransmitter changes and the changes in E2 and LH was determined using the Pearson correlation coefficient as a measure of direction and strength of association. A test consistent with P < 0.05 was considered significant.
Results Group A
Plasma 5HT was highest during the menstrual period (136.25 f 112.3 nmol/L). It decreasedto 66.3 + 37.8 nmol/ L (SD; P < 0.03) during the follicular phase, reached a nadir during ovulation (29.9 + 23.1 nmol/L; P < 0.27), and started to increaseagain during the luteal phase (69.6 + 70.9 nmol/ L; P < 0.14; Fig. 1). Plasma NE during the menstrual phase was 889.52 f 407.15 pmol/L. It decreased during the follicular phase to 474.08 f 314.8 pmol/L (P = 0.0787), rose during ovulation to 876.4 + 629.34 pmol/L (P = 0.09), and peaked during the luteal phase at 1053.25 + 480.5 pmol/L (P < 0.0099 compared to the follicular phase value; Fig. 2). Unless stated otherwise, the P values cited in the above paragraphs represent comparisons to the previous cycle stage. In addition, the multiple comparisons between all stagesare shown in Figs. 1 and 2. Since the results in group A could also have reflected interpersonal variations, a secondgroup of 17 women (group B) was selected. In this group the determinations were performed consecutively in the samewoman during the different phases of the ovulatory cycle, as previously described. The results in this group represent the mean + SD of all determinations performed during the same phase of the ovulatory cycle. It was remarkable that the plasma neurotransmitter levels showed very little variation when meas-
0
L
El
FIG. 1. Mean & SD PPP 5HT levels during the different ovulatory cycle in group A subjects. M, Menstruation; phase; 0, ovulation; L.P., luteal phase.
m
phases of the F.P., follicular
ured in the same patient on the same day or on different days of the samephase of the cycle (results not shown). The values (mean + SD) for PPP-5 HT during the different phases of the cycle as well as the results of the multiple comparison analysis are shown in Fig. 3. From this figure it is seenthat essentially similar results were obtained asin the first group. However, in this group the differences between the phasesof the cycle were statistically significant. When a one-way analysis of variance between PPP 5HT level and phase of the ovulatory cycle was performed, a statistically significant difference between the phases of the ovulatory cycle was achieved (P < 0.001). The phase variation of the ovulatory cycle explains 25% of the variation in plasma 5HT (r’ = 0.25). The difference between the mean values of plasma 5HT in the follicular phase and during ovulation was statistically significant (P < O.OOl), similar to that between ovulation and the luteal phase (P < 0.01). However, no significance was observed between the follicular and luteal phases (Fig. 3). The mean + SD and the results of the multiple comparison analysis of plasma NE during the different phases of the cycle are shown in Fig. 4. The one-way analysis of variance between plasma NE and the phasesof the cycle showed an overall statistically significant difference (P < 0.0001). The changes induced by the different phases of the ovulatory cycle explain 38% of the variation in plasma NE (r’ = 0.38). The increase in plasma NE from the follicular phase to
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926
BLUM
ET AL.
El M
lzl I3
tzl FsP. I3 0
F.R 0
•II L.P.
ml L.P.
n=9
n=17
n=8
m
JCE & M. 1992 VolX.No3
Ei
k17
FIG. 2. Mean + SD PPP NE levels during the different ovulatory cycle in group A subjects. M, Menstruation; phase; 0, ovulation; L.P., luteal phase.
phases of the F.P., follicular
ovulation was significant (P < 0.05), as was the increase from ovulation to the luteal phase (P < 0.01; Fig. 4). Figure 5 shows the mean change over baseline in plasma 5HT, plasma NE, serum EZ,and serum LH during the different phasesof the cycle. From this figure it is seen that the nadir in PPP 5HT during ovulation coincides with the maximal values of serum LH. The overall negative correlation between PPP 5HT and serum LH (-0.07) may indicate that while PPP 5HT increases, serum LH decreases.An overall positive correlation was observed between plasma NE (0.22) and serum Ez. Discussion
The results obtained in group A show that plasma neurotransmitters varied during the menstrual cycle. However, since these values could also have been the result of interpersonal variations, the samemeasurementswere performed consecutively in the samewoman during the different phases of the cycle in 17 subjects (group B) and correlated with hormonal variations. In both groups the neurotransmitter levels varied consistently during the different phasesof the menstrual cycle. Results similar to ours were obtained by Zuspan and Zuspan (20) and Goldstein et al. (22), who found an increase in plasma NE, 2 days before ovulation and a continued increaseafter ovulation, so that the average luteal phase plasma NE level was significantly higher than the
3. Mean + SD PPP 5HT ovulatory cycle in the group ovulation; L.P., luteal phase.
FIG.
levels during B subjects.
the different phases of the F.P., Follicular phase; 0,
follicular phase NE concentration (22). The variations in plasmaNE levels may be attributed to the effects of estrogens on catecholamine degradation (18). It has been shown that estrogens impair NE presynaptic reuptake, compete with catecholaminesfor the degradative enzyme catechol-o-methyltransferase (18), and enhance the rate of monoamineoxidase degradation (25), thereby reducing its activity. Thus, the increase in plasma NE during ovulation and the luteal phase of the cycle observed by us may be explained by an increase in serum estrogen. Another explanation might be linked to the possibility of the plasma levels reflecting processesoccurring in the hypothalamus, such that the above described variations might reflect the hypothalamic NE surge preceding ovulation (7, 26, 27). The variations in PPP 5HT during the menstrual cycle have not been reported previously. Conflicting evidence exists for the role of estrogens in regulation of plasma 5HT
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PLASMA
NEUROTRANSMITTERS
AND
THE
OVULATORY
CYCLE
927
tzl F.P. f
q O ml L.P.
-------LH
:
ml7
i I I
EliI
-NF
iP. FIG. 4. Mean f SD PPP NE levels during ovulatory cycle in the group B subjects. ovulation; L.P., luteal phase.
the different phases of the F.P., Foliicular phase; 0,
levels (28, 29). Theoretically, estrogens should decrease central 5HT by three pathways. 1) Activation of tryptophan 2,3dioxygenase in the liver and the peripheral kynurenine pathway (28) may decrease Trp levels available for 5HT synthesis. B) The second pathway is linked to the previous one; kynurenine may compete with Trp for passage through the bloodbrain barrier (30) and decrease the substrate available for central 5HT synthesis. 3) Estrogens produce a relative absence of pyridoxine, which is an essential cofactor in the activity of L-aromatic acid decarboxylase, one of the enzymes crucial for 5HT synthesis (31, 32). Since the action of PPP 5HT has been shown to parallel phenomena occurring in the brain (12, 14), a decrease in central 5HT might be expected to be accompanied by low PPP 5HT levels. On the other hand, estrogens have also been shown to produce opposite effects by increasing the rate of monoamineoxidase degradation (25) and displacing Trp from its binding sites to plasma albumin (19). In this connection, PPP 5HT has been shown to decrease after ovariectomy (29) and menopause (33). In our two groups of patients we observed large differences in PPP 5HT levels between the ovulatory and luteal phases,
i,
LI.P.
FIG. 5. The correlation between PPP 5HT and NE variations and E, and LH during the different phases of the ovulatory cycle. The results are expressed as the mean change over baseline. F.P., Follicular phase; 0, ovulation; L.P., luteal phase.
when similar estrogen levels were observed. No consensus has been reached in the literature for the role of 5HT in GnRH and gonadotropin secretion (8, 34). However, the most convincing evidence has been adduced for an inhibitory role of 5HT on GnRH and gonadotropin secretion (35,36). Ovulation occurs only when this inhibitory influence disappears. This latter theory might be in concordance with the significant drop in the PPP 5HT level during ovulation and the significant inverse correlation between PPP 5HT and serum LH. However, a peripheral effect of LH on PPP 5HT cannot be ruled out by the present study. High plasma NE levels during menstruation have already been reported by Feichtinger et al. (21). In our group A patients similar high levels of plasma NE and 5HT were observed. It is possible that the high levels of PPP 5HT and NE during this period are explained by the stress produced by menstrual pains. The increased stress levels might produce a secondary increase in adrenal catecholamine output as a result of the increase in glucocorticoid levels (37). However, this mechanism does not explain the increase in PPP 5HT due to glucocorticoids decreasing 5HT levels by diverting its
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928
BLUM
precursor to the kynurenine pathway (38). Another more plausible explanation might be linked to the peripheral release of 5HT and NE from the platelets during menstruation. In conclusion, plasma neurotransmitter levels have been shown to vary predictably during the menstrual cycle. Plasma NE levels are lowest during the follicular phase and highest during the luteal phase and menstruation; plasma 5HT levels are highest during menstruation and lowest during ovulation. These findings should be taken into consideration in the design and analysis of studies that use PPP 5HT and NE as indicators of serotonergic and adrenergic activities in different diseases. At this stage there is no clear-cut evidence linking the changes in plasma neurotransmitter profile, observed by us, to hormonal, neuronal, or central neurotransmitter changes. However, several lines of evidence indicate that PPP 5HT and plasma NE reflect central processes. Results from Artigas’s laboratory showed the existence of a freely circulating pool of 5HT in the plasma different from that in the platelets (39,40). This pool reacts differently to chronic drug treatment than does the platelet pool. Thus, chronic treatment with lithium salts in bipolar patients increased PPP 5HT levels, while leaving platelet 5HT concentrations unchanged (14). Results obtained in our laboratory showed that changes in meal composition producing changes in the availability of tryptophan (the precursor of 5HT synthesis) for transport through the blood-brain barrier induced changes in PPP 5HT similar to those reported in the brain (12,41). Taken together these results indicate that PPP 5HT may be a useful peripheral model to study brain processes mediated by 5HT. Plasma NE has already been shown to reflect neuronally occurring processes (23). These facts concur with the results reported by Goldstein et al. (22), who showed an increase in plasma NE before ovulation, in suggesting that changes in the plasma neurotransmitter profile reflect hypothalamic processes linked to ovulation. Thus, the increase in EZ levels before ovulation that produces an increase in hypothalamic NE, which mediates the GnRH surge (7), seems to be reflected in the plasma levels. However, much more work is needed to explore these questions. Our findings might serve to elucidate different pathological states linked to the menstrual cycle. In addition, it is possible that in the future these findings might help women suffering from mood disorders linked to the menstrual cycle or women experiencing different anovulatory states. References 1. Meltzer HY, Lowy MT. 1987 The serotonin hypothesis of depression. In: Meltzer HY, ed. Psychopharmacology: third generation of progress. New York: Raven Press; pp 513-26. 2. Schildkraut JJ. 1965 The catecholamine hypothesis of affective disorders: a review of supportive evidence. Am J Psychiatry. 122:509-22. 3. Campese VM, De Quattro V. 1989 Functional components of the sympathetic nervous system: regulation of organ systems. In: De Groot LJ, ed. Endocrinology, 2nd ed, Philadelphia: Saunders; pp 1738-56. 4. Gallo RV. 1980 Neuroendocrine regulation of pulsatile luteinizing hormone in the rat. Neuroendocrinology. 30:122-31. 5. Kreiger HB, Krieger DT. 1970 Chemical stimulation of the brain:
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