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J Appl Physiol 82: 364-370, 1997;
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Journal of Applied Physiology
Vol. 82, No. 1, pp. 364-370, January 1997

RAPID COMMUNICATION

Plasma 2-hydroxycatecholestrogen responses to acute submaximal and maximal exercise in untrained women

Carl De Crée1, Peter Ball2, Bärbel Seidlitz2, Gerrit Van Kranenburg3, Peter Geurten3, and Hans A. Keizer3

Interuniversity Project on Reproductive Endocrinology in Women and Exercise: 1 Department of Applied and Experimental Reproductive Endocrinology, The Institute for Gyneco-Endocrinological Research, Leuven 3, Belgium; 2 Department of Biochemical and Clinical Endocrinology, Medical University of Lübeck, D-23538 Lübeck, Germany; and 3 Department of Movement Sciences, Faculty of Health Sciences, University of Maastricht, NL-6200 MD Maastricht, The Netherlands

ABSTRACT
INTRODUCTION
SUBJECTS AND METHODS
RESULTS
DISCUSSION
ACKNOWLEDGEMENTS
FOOTNOTES
REFERENCES

ABSTRACT

De Crée, Carl, Peter Ball, Bärbel Seidlitz, Gerrit Van Kranenburg, Peter Geurten, and Hans A. Keizer. Plasma 2-hydroxycatecholestrogen responses to acute submaximal and maximal exercise in untrained women. J. Appl. Physiol. 82(1): 364-370, 1997.---Exercise-induced menstrual problems are accompanied by an increase in catecholestrogen (CE) formation. It has been hypothesized that hypoestrogenemia may be secondary to an increased turnover from estrogens to CE, which then may disrupt luteinizing hormone release. In addition, the strong affinity of CE for the catecholamine-deactivating enzyme catechol-O-methyltransferase (COMT) has led to speculations about their possible role in safeguarding norepinephrine from premature decomposition during exercise. We investigated whether acute exercise on a cycle ergometer produces any changes in CE homeostasis. Nine untrained eumenorrheic women (body fat, 24.8 ± 3.1%) volunteered for this study. Baseline plasma CE averages for total 2-hydroxyestrogens (2-OHE) were 218 ± 29 (SE) pg/ml during the follicular phase (FPh) and 420 ± 58 pg/ml during the luteal phase (LPh). 2-Methoxyestrogens (2-MeOE) measured 257 ± 17 pg/ml in the FPh and 339 ± 39 pg/ml in the LPh. During incremental exercise, total estrogens (E) increased, but 2-OHE and 2-MeOE levels did not significantly change in either phase. The 2-OHE/E ratio (measure of CE turnover) decreased during exercise in both menstrual phases, whereas the 2-MeOE/2-OHE ratio (correlates with COMT activity) did not significantly change. These findings suggest that there is insufficient evidence to conclude that brief incremental exercise in untrained eumenorrheic females acutely produces increased CE formation.

amenorrhea; catecholamines; catechol-O-methyltransferase; estrogens; menstrual cycle

INTRODUCTION

PHYSICAL EXERCISE IN WOMEN provokes important changes in plasma concentrations of sex hormones (17). Changes in menstrual and bone status have been identified as the most prominent effects of long-term strenuous exercise (7). However, most of these phenomena are still poorly understood. Diet, weight loss, relative hyperprolactinemia, and percentage of body fat have all been suggested as mediating factors (20). To date, there is no consensus as to the precise mechanisms involved. The only consensus applies to the hypoestrogenic status and disturbed gonadotropin oscillator, which are generally observed in female athletes with exercise-related menstrual irregularities (20). Previously, most studies have examined basic reproductive hormones, without looking much further into the actual hormonal metabolism. In one of the few exceptions, Snow and co-workers (29) examined estrogen metabolism. By measuring the converting enzyme, these authors found that a group of oarswomen with menstrual problems exhibited a significantly higher C2-hydroxylase oxidation than did a group of eumenorrheic oarswomen enrolled in the same training practice. Moreover, the extent of C2-hydroxylase activity was positively correlated with leanness. These findings suggested that exercise promotes a shift in estrogen metabolism from 16alpha - to C2-hydroxylation. This would be in agreement with earlier studies from Russel et al. (26), who found the highest circulating levels of C2-hydroxylated estrogens in the most vigorously training group of swimmers, who also happened to be oligomenorrheic.

The C2-hydroxylation of estrogens leads to the formation of the 2-hydroxyestrogens (2-OHE) and their monomethylethers, the 2-methoxyestrogens (2-MeOE; 6). Both groups of estrogen metabolites are part of the so-called catecholestrogens (CE). It has been demonstrated that the formation of 2-hydroxy and 2-methoxy CE represents a major metabolic pathway for estrogen metabolism (5). Because of their high instability and the laborious detection methods required, research into CE has been limited. However, it has been shown that CE have the potential to control luteinizing hormone (LH) release. "Having the potential," here literally means that sometimes they do and sometimes they don't control LH secretion; when they do, their effect may be either stimulatory or inhibitory. Their precise action appears to depend strongly on the type of CE, the richness of the steroid environment, and the individual's specific brain area involved in CE formation (21). Mainly because of these restrictions, previous evidence in support of a role for CE in modulation of the reproductive axis is controversial.

For example, Adashi et al. (2) observed in hypogonadal women that 2-hydroxyestrone only exerted effects after prior estrogen priming. They found that administration of an infusion of the C2-hydroxylated metabolite of estrone, 2-hydroxyestrone, produced a small and rapid rise in LH, followed by a fall in LH levels lasting for hours. These results were later confirmed by Schinfeld et al. (27). With the use of the C2-hydroxylated metabolite of estradiol (E2), 2-hydroxyestradiol, administered by bolus doses to both premenopausal and postmenopausal women, Miyabo and co-workers (24) failed to observe any effects on LH release by this CE. However, infusion to hypogonadal females resulted in significant suppressive effects on LH release, but only after prior estrogen priming (1). Apart from the methodological differences already mentioned above, Parvizzi and Ellendorf (25) may have offered an explanation for the lack of consensus in findings. They showed in pigs that microinjections of 2-hydroxyestradiol into the ventromedial nucleus of the hypothalamus suppress LH, whereas microinjections of the same CE into the preoptic area medialis of the hypothalamus results in a positive-feedback action on gonadotropin release. Using similar techniques, these authors also unveiled the importance of an intracerebral steady state of 2-hydroxyestrone essential to produce an ovulatory LH surge.

Despite the conflicting findings of some studies that have examined the effects of CE on gonadotropin release, even less seems evident about the mechanism that actually triggers the shift in estrogen metabolism toward C2 hydroxylation. For exercise specifically, one might wonder whether the amount or intensity of acute exercise is of any importance for stimulating such a shift in metabolic pathway? Or is the extent of C2-oxidation merely a long-term effect that entirely depends on some body fat threshold, as apparently suggested by Snow et al. (29)?

In 1990, it was hypothesized that a complex feedback system involving CE underpins exercise-related menstrual problems (9). This theory linked in a cohesive way recent findings on the modulation of neuroendocrine pulsatile regulation of gonadotropins with menstrual phenomena observed in women athletes. It offered an explanation as to why a shift toward C2-hydroxylation might occur in response to exercise. It was suggested that there is a very specific physiological reason for this phenomenon. Indeed, 2-OHE not only exhibit an LH-mediating effect but may also facilitate the involvement of estrogens in energy metabolism. Biochemically, CE are substances with both catecholamine (CA) and estrogen capabilities. They strongly inhibit the enzymatic methylation and biological inactivation of the neurotransmitters epinephrine (Epi) and norepinephrine (NE) by catechol-O-methyltransferase (COMT; 6). In other words, the hypothesized physiological role of CE during acute exercise would be to safeguard CA from premature decomposition by increasingly competing for COMT.

The rationale for the present study is to offer a first and partial investigation of the above-mentioned hypothesis, which has attempted to link the previously reported responses of CE after chronic exercise with its postulated role during acute exercise. At present, there is a complete lack of knowledge about acute exercise-induced changes in plasma CE. For example, it is unclear whether acutely elevated CE levels are accompanied by simultaneous decreases in circulating estrogens. The data from Russel et al. (26) do not resolve this question, because one might argue that the hypoestrogenemia in their subjects could have also resulted from chronically reduced gonadotropin support. The purpose of this study was to collect elementary information on the resting levels and acute exercise-induced plasma responses of 2-hydroxycatecholestrogens in young, untrained, eumenorrheic women. In addition, we wondered what we could learn about CE formation and activity, as far as it is possible to make a valid estimation about these parameters from using simple, indirect measuring techniques (turnover ratios) instead of using radioactive tracers in healthy subjects.

In the present study, we have also limited ourselves to investigate specifically the above-mentioned CE, although fully realizing that many CE other than the 2-OHE exist. The main argument in favor of our choice is that the few other studies that have paid any attention to the link between exercise and CE studied the same components. In addition, the 4-OHE, for example, are even less stable and circulate in amounts closer to minimal detection rates.

SUBJECTS AND METHODS

Subjects

Nine nulliparous, eumenorrheic, healthy, untrained Caucasian women [age 20.4 ± 1.3 (mean ± SD) yr; height 172.0 ± 4.7 cm; body mass 61.4 ± 4.8 kg; %body fat 24.8 ± 3.1%; age of menarche 13.2 ± 1.3 yr; menstrual cycle length 28.9 ± 2.9 days] provided written informed consent and were recruited. Each subject filled in a questionnaire to identify her athletic activities as well as menstrual and gynecological history. All subjects had to fulfill various qualifications. Menstrual cycles had to be normal for at least a year prior to the study; they had to be free of any gynecological disorder or abnormal liver, renal, and thyroid functioning; and they were not allowed to take any drugs or contraceptives. Subjects kept a menstrual diary and were given a set of Ovusticks (Samenwerkende Apothekers, The Netherlands) to determine ovulation from analysis of morning urine. All subjects had normal ovulatory menstrual cycles, as evidenced by plasma progesterone (P4) levels (data not shown), basal body temperature, and Ovusticks. Nutrient and daily energy intake did not differ among the subjects when menstrual phases were compared. Percentage body fat was estimated from skinfold measurements (12), using a Harpenden calliper (Holtain, Crymych, UK). The subjects acted as their own controls.

Experimental Design

All tests were performed on an electrically braked cycle ergometer (Lode, Groningen, The Netherlands) with continuous electrocardiogram surveillance. Two standardized incremental exercise tests were undertaken in each menstrual phase: one in the follicular phase (FPh) between days 7 and 10, and one in the luteal phase (LPh) between days 23 and 25. Half an hour before the test, a venous indwelling 21-gauge Teflon Quick catheter (Travenol Laboratories, Deerfield, IL) was inserted into an antecubital vein for blood collection and subsequent determination of total protein and hormone concentrations. A stopcock was attached to the catheter, and circulation was assured by injecting 1 ml of sterile saline periodically.

The exercise protocol is illustrated in Fig. 1. During a 5-min period (t-5 and t0), the subject was placed on the cycle ergometer. After a 2-min warm-up period at 25 W, workload was established for 4 min at 50 W. Workload was increased by increments of 50 W each 4-min interval up to 150 W. After subjects cycled for 4 min at 150 W, which coincided with submaximal intensity (plasma lactate concentration of 2.0-3.0 mM), the workload was then lowered for 2 min to 50 W to allow collection of blood samples (tsubmax). Workload was then raised again for 1 min at 150 W. Each following minute, the workload was increased by 25 W, until the subject was unable to continue exercise, despite vocal encouragement (tmax). Intensity was lowered to 50 W for 10 min of recovery.

Fig. 1. Schematic overview of exercise protocol. VO2 max, maximum O2 uptake.
[View Larger Version of this Image (16K GIF file)]

During exercise, the subject breathed through a mouthpiece attached to a turbine device. Expired air was collected and analyzed breath by breath by using an Oxycon beta  (Mijnhardt-Jäger, Bunnik, The Netherlands) automated device, which was calibrated before and after testing, using gases of known concentration, volume, and flow rates. Ventilation, oxygen intake, and carbon dioxide expiration were determined per unit of time.

Blood Hormone and Biochemical Analysis

Blood for hormone analysis was drawn into disposable 20-ml syringes both at rest and during exercise at intensities equivalent to submaximal and maximal exercise levels (100% VO2). Blood samples were collected in precooled lyophilized EDTA glass tubes stored in an ice bath at 2°C. Blood samples for estrogen determination were immediately centrifuged at 2,000 g for 10 min at 4°C, deep frozen by liquid nitrogen at -196°C, and stored at -80°C until assayed. Plasma LH was measured by an immunoradiometric assay (Serono, Geneva, Switzerland). The cross reactivity with thyroid-stimulating hormone was 1.6%, and <0.1% with human chorionic gonadotropin and follicle-stimulating hormone. The sensitivity was 0.4 mIU/ml, and the intra-assay coefficient of variation was <15.5% at 1 mIU/ml, below 8% at 1.5 mIU/ml, and <5% at 10 mIU/ml. Plasma P4 was measured after ether extraction by commercially available kits from Radio-isotopen Service, Würlingen, Switzerland [interassay coefficient of variation (cvb) 7.2%, minimal detectable concentration 0.13 ng/ml]. Plasma CA concentrations of NE, Epi, and dopamine (DA) were determined by high-performance liquid chromatography (HPLC), using a Waters WISP 710B injector (Millipore, Milford, MA), a Hitachi L 6200A pump (Hitachi, Tokyo, Japan), an electrochemical detector (ESA Coulochem II, Interscience, Bedford, MA), and an Alltech 9781 column, connected to an IBM-compatible computer and Interface D-6000 with Hitachi HPLC-Manager software. Interassay coefficients were 4.5% for NE and <11.8% for Epi and DA. All standards used in the hormone analysis were assayed against Medical Research Council preparations. The intra-assay variability (cvi) was <10%; the interassay variability as determined from pool plasma was 13.3%; and cross reactivity was not significant.

Blood for determination of 2-hydroxy CE and their 2-hydroxy monomethylethers was prepared as follows. After centrifugation as described above, 2 ml of plasma were pipetted in 5-ml polyethylene Eppendorf cups in which 1 ml of a 3% aqueous ascorbic acid solution had been added to prevent oxidative decomposition. Preparations were then stored at -80°C until assaying, as described above. Under these circumstances, CE remain stable for numerous months (3). CE were analyzed by radioimmunoassay. Free, i.e., unconjugated CE, in plasma are near or below the detection limit of normal CE assay procedures (13). Therefore, for the purpose of this paper, and for the sake of validity and particularly accuracy, we have chosen to determine the unconjugated and conjugated fractions (the latter after acid hydrolysis) in one assay procedure. The assay to measure plasma CE levels involved the following five steps.

Acid hydrolysis. First, 0.8 ml of ascorbic acid-stabilized plasma was added to 630 µl of solution A (2.1 g potassium iodide, 24 ml H2O, 10.57 ml concentrated HCl, and 28 µl 1% Na2S2O5) and heated for 1 h at 100°C. After cooling, 150 µl 1% Na2S2O5 and 1,000-1,500 disintegrations/min [6,7] tritiated tracer of 2-hydroxyestradiol (2-OHE2), 2-methoxyestradiol (2-MeOE2), and E2 were added and vortexed for 30 s. Radioactivity was checked, and the aqueous mixture was extracted once with 3 ml and twice with 2 ml of ether/ethyl acetate (3:1). The combined extracts were washed once with 1.5 ml of solution B (ascorbic acid buffer, pH 10.2; 100 ml of a stock solution of 45 g NaHCO3, 615 ml H2O, 150 ml methanol, and 9 mg NaOH freshly prepared before use by the addition of 5 g of ascorbic acid and titration to pH 10.2 by the use of 10 N NaOH), followed by 1.5 ml of water and then evaporated to dryness under N2 at 38°C (water bath).

Reduction. The residue was redissolved in 0.5 ml of methanol, and every 30 min, 200 µl of solution C (75 mg NaBH4, 50 ml H2O, and 150 µl of 0.1 N NaOH) were added. The reduction was stopped by the addition of concentrated CH3COOH. Excess methanol was removed at 37°C under N2 and then filled up with water to 1 ml before extracting twice with 2 ml of benzene-ethylacetate (1:1).

Borate distribution. The above extract was washed twice with solution D (borate buffer: 9.3 g boric acid, 13.2 g ascorbic acid, 195 ml 1 N NaOH, 60 ml methanol, 45 ml H2O, titrated with 10 N NaOH to pH 10.7), leading to a separation of catecholic (aqueous phase) and nonphenolic (organic phase) steroids. The organic phase was subsequently washed once with 3 ml of 10% CH3COOH, stabilized by the addition of 100 µl of solution E (1% ascorbic acid and 10% CH3COOH in ethanol), evaporated to dryness at 37°C under N2, and redissolved in 0.5 ml of solution F (benzene-methanol, 95:5, vol/vol). The aqueous phase was acidified with 0.4 ml of concentrated HCl before extracting twice with 3 ml of benzene-ethyl acetate (1:1, vol/vol). The combined extracts were subsequently washed once with 3 ml of 10% of CH3COOH, stabilized by the addition of 100 µl of solution D, evaporated to dryness at 38°C under N2, and redissolved in 0.5 of solution G (benzene-methanol, 90:10, vol/vol).

LH20 columns. Separation on LH20 columns was accomplished by using solutions F (E2 and 2-MeOE2) or G (2- and 4-OHE2). The appropriate fractions of the elutes were taken to dryness under N2 and redissolved in 1,000 µl (E2 + 2-MeOE2) of solution E for storage.

Assay procedure. All assays made use of highly specific antibodies raised in rabbits. The synthesis, production, and characteristics of the antisera have been detailed previously (3, 14). The 2-OHE2 antisera for the determination of total CE were chosen for their ability to exhibit a high cross reactivity for D-ring-modified steroids, namely 2-OHE2, without showing any significant cross reactivity with A-ring-modified estrogens, such as 4-OHE2 or the monomethylethers of 2- and 4-OHE2 (Table 1).

Table 1. Cross-reactivity of steroids and catecholamines with catecholestrogen antiserum

Steroid 2-Hydroxyestrone Antiserum, %  2-Methoxyestrone Antiserum, % 
C18 steroids
  2-Hydroxyestrone 100 0.3
  2-Hydroxyestradiol 26 <0.1
  2-Methoxyestrone <0.1 100
  2-Methoxyestradiol <0.1 44
  2-Hydroxyestriol 4.5 <0.1
  4-Hydroxyestrone 0.5 <0.1
  4-Hydroxyestradiol 0.1 <0.1
  Estrone 0.8 0.3
  Estradiol 0.2 <0.1
  Estriol <0.1 <0.1
C19 steroids, androstane group <0.1 <0.1
C21 steroids, pregnane group <0.1 <0.1
Catecholamines <0.1 <0.1

The plasma CE values in this paper are reported as total 2-OHE and total 2-hydroxyestrogen 2-monomethylethers or 2-methoxyestrogens (2-MeOE). The term "total" refers to the sum of the unconjugated and conjugated fractions of the estrone- and estradiol-derived CE metabolite together. Specifically, total 2-OHE consists of 2-OHE1 + 2-OHE2; and total 2-MeOE includes 2-MeOE1 + 2-MeOE2. Therefore, to have a comparable measure of the subjects' plasma primary estrogen levels, we also determined plasma total estrogens (E), instead of the separated unconjugated E2 fraction, which is most commonly used in the literature on hormones and exercise. The term total estrogens, as used in this paper, refers to the sum of unconjugated and conjugated estrone and estradiol only. The term here includes neither the 16alpha -hydroxyestrogen (16alpha -hydroxyestrone and estriol) nor the 15alpha -hydroxyestriol (estetrol) metabolites. For the purpose of the present study, it was useful to have an idea of actual CE formation (turnover from conventional estrogens) and activity (competition for COMT). To avoid the use of radioactively labeled substances in healthy young subjects, we used the 2-OHE/E ratio as a measure of CE formation, and the 2-MeOE/2-OHE ratio as a measure of CE activity or O-methylation, as described previously (10). Coefficients of variation (cvi) are <10% for E, 2-OHE, and 2-MeOE. The interassay precision, as determined from pool plasma, resulted in a coefficient of variation (cvb) of 13.3% for E, 14.1% for 2-OHE, and 7.2% for 2-MeOE. The lower limit of detection for all CE assays was ~6 pg/ml.

Total protein was determined by the biuret method. The biuret reagent was obtained from Hoffman LaRoche (no. 1010083; in mM: 200 K-Na tartrate, 120 CuSO4, 100 KJ, 2 NaOH). Hemoglobin was determined spectrophotochemically with the hemoglobin cyanide method by using a Unicam model SP-600. Hematocrit was determined by the microcentrifuge method, and blood lactate was determined with an electrical-chemical-enzymatic method, using a semiautomatic lactate analyzer (Lactate Analyzer 640, Kontron, Zurich, Switzerland). Results were controlled for changes in plasma volume.

Statistical Analysis

Comparisons between different menstrual phases were made by a two-way mixed model analysis of variance for groups, with repeated measures on both factors. Hormonal responses were analyzed according to 1) contrasts of serial blood sampling times within each of the two exercise sessions, 2) the two exercise sessions spreading over follicular and luteal phases, and 3) interactions between sampling times and exercise sessions. The level of significance for individual contrasts within each of the subjects was adjusted to limit the experimental error rate to a maximum of 5%. Statistical significance was reevaluated after Bonferroni correction. The ratios of the steroid responses were evaluated by analyzing the magnitude of absolute and relative changes during submaximal and maximal load from rest values. Phase, group, phase group (interactive), and subject effects were evaluated independently by maximum likelihood procedures.

RESULTS

Physiological Characteristics

Physiological characteristics are illustrated in Table 2. The maximal physical working capacity (MPWC) was higher during the LPh, but the mean VO2 max was significantly lower (41.5 ± 1.4 vs. 45.7 ± 1.5 ml · kg-1 · min-1, LPh vs. FPh, respectively; P < 0.05).

Table 2. Physiological characteristics during follicular and luteal phases

Variable Follicular Luteal
Pmax, W 241 ± 13.5  250 ± 10.4 
HRmax, beats/min 188 ± 2.6  184 ± 4.3 
 VO2 submax, ml · kg-1 · min-1 34.5 ± 2.3  30.6 ± 1.6*
 VO2 max, ml · min-1 · min-1 45.7 ± 1.5  41.5 ± 1.4*
Values are means ± SE. Pmax, maximum pressure; HRmax, maximum heart rate; VO2 max, maximum oxygen uptake. * Significantly different from follicular phase values, P < 0.05.

Hormonal Responses

Hormonal responses are shown in Table 3. Mean levels of E progressively increased during exercise in the LPh (t0, 3,899 ± 1,134 pg/ml; tsubmax, 4,110 ± 1,274 pg/ml; tmax, 4,617 ± 1,356 pg/ml). However, the observed acute exercise-induced increases in plasma estrogen concentrations in both menstrual phases only reached significance (P < 0.05) at maximal intensity (FPh, +12%; LPh, +18% at tmax; Fig. 2). Mean plasma total CE during baseline conditions in women were significantly different (P < 0.05) between menstrual phases. For 2-OHE, we measured 218 ± 29 pg/ml during the FPh and 420 ± 58 pg/ml during the LPh. For 2-MeOE, we found 257 ± 17 pg/ml during the FPh and 339 ± 32 pg/ml during the LPh. During incremental exercise, 2-OHE and 2-MeOE did not significantly increase in either menstrual phase. CE formation, as expressed by the 2-OHE/E ratio, at maximal intensity decreased by 6% from baseline in the FPh and by 18% in the LPh. CE activity, meaning the amount of O-methylated CE as calculated from the 2-MeOE/2-OHE ratio, did not significantly change during incremental exercise.

Table 3. Plasma estrogen and catecholestrogen responses to acute incremental exercise

Hormone Exercise Intensity
t0
tsubmax
tmax
Follicular Luteal Follicular Luteal Follicular Luteal
E, pg/ml 1,656 ± 431  3,899 ± 1,134dagger 1,646 ± 419  4,080 ± 1,274dagger 1,860 ± 473* 4,617 ± 1,356*dagger
2-OHE, pg/ml 218 ± 29  420 ± 58dagger 234 ± 28  422 ± 53dagger 231 ± 29  422 ± 57dagger
2-MeOE, pg/ml 257 ± 17  339 ± 32dagger 257 ± 20  339 ± 16dagger 275 ± 20  354 ± 34dagger
2-OHE/E 0.17 ± 0.03  0.17 ± 0.04  0.18 ± 0.03  0.16 ± 0.04  0.16 ± 0.03  0.14 ± 0.04 
2-MeOE/2-OHE 1.27 ± 0.09  0.88 ± 0.07dagger 1.28 ± 0.09  0.93 ± 0.09dagger 1.28 ± 0.11  0.91 ± 0.08dagger
Values are means ± SE. t0, time 0; tmax, maximal intensity; tsubmax, submaximal intensity; E, estrogen; 2-OHE, 2-hydroxyestrogens; 2-MeOE, 2-methoxyestrogens; 2-OHE/E, ratio of 2-OHE to E; 2-MeOE/2-OHE, ratio of 2-MeOE to 2-OHE. * Plasma concentration significantly different from resting values at t0, P < 0.05; dagger plasma concentration or calculated ratio significantly different between menstrual phases, P < 0.05.

Fig. 2. Mean net adjusted differences (%) from baseline of plasma estrogen and catecholestrogen responses to acute exercise. Baseline of each menstrual phase = 0%. * Response significantly different from baseline (P < 0.05); dagger  response significantly different between phases (P < 0.05).
[View Larger Version of this Image (24K GIF file)]

Plasma CA levels significantly rose in response to incremental exercise (Table 4). Circulating concentrations of plasma CA showed differences in absolute value between menstrual phases. At submaximal exercise intensity, both NE and Epi were higher during the FPh, whereas at exhaustion, NE and Epi were significantly higher (P < 0.05) during the LPh.

Table 4. LH and catecholamine responses to incremental exercise and training

Phase Exercise Level Norepinephrine, pg/ml Epinephrine, pg/ml LH, mIU/ml
Follicular Baseline (t0) 178 ± 51  22.0 ± 9.8  3.5 ± 1.5 
Luteal Baseline (t0) 167 ± 146  43.5 ± 25.3  4.3 ± 4.5 
Follicular Submaximal (tsubmax) 763 ± 781*dagger 76.8 ± 92.1*dagger
Luteal Submaximal (tsubmax) 338 ± 278*dagger 65.8 ± 37.5*dagger
Follicular Maximal (tmax) 1,411 ± 867*dagger 292.9 ± 299.7*dagger
Luteal Maximal (tmax) 2,215 ± 1,524*dagger 453.8 ± 379.8*dagger
Values are means ± SE. LH, luteinizing hormone. * Significantly different from baseline value; dagger significantly different between menstrual phases; P < 0.05.

DISCUSSION

Findings on Menstrual Cycle-Related Physiological Parameters During Acute Exercise

VO2 max was significantly lower during the LPh than during the FPh. Data in the literature are confusing about differences in exercise performance between menstrual phases. Some studies have found an enhanced performance in either the FPh (8) or the LPh (17), whereas others found no differences between phases (18). Neither from published data nor from the findings of the present study is it possible to provide an explanation for these discrepancies beyond speculation. Negligence in accurately determining the menstrual phase of the subjects and lack of rigor in exercise protocols have been assumed to account for most of the differences in findings reported in the literature (20). Furthermore, it has been suggested that the higher circulating progesterone levels in LPh would increase ventilation at rest, resulting in maximal ventilation attained at a relatively lower intensity during the LPh (28).

Estrogens and Acute Exercise

Plasma E concentrations at submaximal and maximal intensity exercise were increased in both menstrual phases, compared with baseline levels. This finding is in agreement with other studies (18, 20), although these mainly measured unconjugated E2. The acute exercise-induced increments in circulating estrogen have been shown before to result from a decreased metabolic clearance rate (MCR) during acute exercise (19). In one subject, we found E concentrations up to >15,000 pg/ml. A possible explanation is that the subject had a very high transferase activity, with a high percentage of conjugated estrogen.

CE and Acute Exercise

Data on CE during acute physical exercise are not available in the literature. In the present study, no significant changes in either plasma 2-OHE or 2-MeOE were found during submaximal or maximal exercise, compared with resting values. Although CE formation, expressed by the 2-hydroxy CE to conventional estrogen ratio, decreased during acute exercise in both menstrual phases, the clear increase in E might have easily accounted for this difference. This obviously questions the validity of the 2-OHE/E ratio. The tremendous difference in MCR of estrogens and CE (at rest, averaging 20,000-40,000 l/day, or ~20-50 times higher than the MCR of E2) is also likely to bias the outcome of this ratio. However, unless one administered radioactively labeled compounds to healthy subjects, there is simply no better alternative to obtain an indication of CE formation. The ratio of O-methylated CE to non-O-methylated CE is assumed to correlate with the actual CE activity (10). This ratio showed only small increases up to 6% during the LPh. Nevertheless, if the simultaneous decrease in 2-OHE/E (up to -18%) could chiefly be attributed to a lower formation of CE, then this would mean that the percentage CE that becomes O-methylated increases, despite less CE being formed. In other words, it would be evidence that acute exercise in eumenorrheic women indeed stimulates CE to compete increasingly for COMT. Unfortunately, because of the limitations detailed above, it is impossible to infer this from the present results.

Menstrual-Phase Differences

Baseline circulating plasma levels of estrogens and CE were higher during the LPh than during the FPh. This is in agreement with previous studies (6, 13). Responses to acute exercise were not significantly different between phases, except for E which, at maximal intensity increased to a higher extent during the LPh. However, when data were reanalyzed as percent increases from baseline, there were no significant differences between phases. Baseline 2-OHE/E and 2-MeOE/2-OHE ratios were higher in the FPh than in the LPh. Although it could be argued again that the difference in 2-OHE/E ratio is a consequence of pronounced differences in FPh and LPh conventional estrogen levels, rather than being caused by differences in CE formation, it will not explain the observed phase differences in 2-MeOE/2-OHE ratio. In our opinion, these phase differences in CE metabolism behavior suggest that the formation and activity of CE in the untrained female is less pronounced during the LPh.

Possible Implications for Menstrual Cycle Irregularities

Studies in nonathletes have shown that certain CE are capable of suppressing the gonadotropin pulse oscillator. In many previous studies, it has been postulated that CE play an important role in the regulation of the menstrual cycle (4). It has also been shown that CE appear to be involved in a number of very different mechanisms, which all, nevertheless, have been related to causing menstrual problems. For example, CE have also been found to control prolactin secretion (15, 23) and to stimulate the production of the luteolytic uterine prostaglandin F2alpha (PGF2alpha ; 16). PGF2alpha has been shown previously to increase with acute physical exercise (11). Premature corpus luteum destruction observed in women with exercise-induced menstrual problems has been linked to enhanced PGF2alpha formation (9).

The previously reported (29) significantly higher baseline 2-hydroxylase activity in oligomenorrheic athletes strongly suggests that CE are somehow involved in the etiology of exercise-related menstrual problems. Yet it remains difficult to reconcile better established hypotheses with the speculation that disturbance of gonadotropin secretion is an effect secondary to hypoestrogenemia resulting from an increased formation of CE. There is still more support in favor of hypoestrogenemia in female athletes being secondary to a central suppression of the GnRH pulse generator. We believe, however, that this previous evidence does not entirely discredit the existence of a complex feedback system, whereby gonadotropin secretion and the balance estrogen/CE formation mediate each other in a reciprocal way.

In conclusion, the results of the present study show that acute exercise does not alter the circulatory levels of 2-OHE and the O-methylated product 2-MeOE. In the present study, CE formation (as expressed by the 2-OHE/E ratio) and CE activity (as expressed by the 2-MeOE/2-OHE ratio) were lower during the LPh compared with the FPh, which may suggest an increased O-methylation and a more active involvement of CE during exercise in the FPh. However, the absence of more convincing evidence obtained by radioactive tracer methods, the limitations of these ratios, and the low significance of our findings, leave the role of CE during acute exercise open to further debate. Accordingly, the results of the present study do not allow us to make any further deductions with regard to the previously postulated role of CE in safeguarding CA availability.

ACKNOWLEDGEMENTS

We thank all the women who kindly participated in this study. We gratefully acknowledge the help of Dr. A. Vermeulen of the Department of Endocrinology and Metabolism of the State University of Ghent and the help of Dr. M. Ostyn of the Institute of Physical Education of the Catholic University of Leuven, Belgium, both for their scientific advice as to the experimental design and for preparation of this study. Y. Janssen, M. Van Der Heyden, and K. Mannheimer provided excellent technical skills. Also, we thank A. Bialkowska for the computer graph and tables, and K. Hibler, G. Hibler, and E. M. Winter for proofreading the manuscript and for their critical comments.

FOOTNOTES

Address for reprint requests: C. De Crée, Dept. of Applied and Experimental Reproductive Endocrinology, The Institute for Gyneco-Endocrinological Research, PO Box 134, B-3000 Leuven 3, Belgium.

Received 22 July 1996; accepted in final form 28 October 1996.

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C. De Cree, P. Ball, B. Seidlitz, G. Van Kranenburg, P. Geurten, and H. A. Keizer
Effects of a training program on resting plasma 2-hydroxycatecholestrogen levels in eumenorrheic women
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