Zinc and copper levels in premenstrual syndrome*

Transcription

1 FERTILITY AND STERILITY Copyright 1994 The American Fertility Society Printed on acid-free paper in U. S. A. Zinc and copper levels in premenstrual syndrome* C. James Chuong, M.D., M.P.H.t Earl B. Dawson, Ph.D.t Department of Obstetrics and Gynecology, Baylor College of Medicine, Houston, and University of Texas Medical Branch, Galveston, Texas Objective: To determine whether changes in peripheral zinc and copper levels are associated with symptoms of premenstrual syndrome (PMS). Design: Ten PMS patients and 10 controls gave blood at 2- or 3-day intervals through three menstrual cycles. Serum zinc and copper were measured by flameless atomic absorption spectrophotometry. Results: In the controls, zinc values were not significantly different between the follicular and the luteal phases. In the patients, the values were significantly lower during the luteal phase than during the follicular phase. Lower levels of zinc during the luteal phase in PMS patients, compared with the controls, were noted. Copper levels were noted to be higher during the luteal phase in PMS patients compared with the controls. Because copper competes with zinc for intestinal absorption and serum protein binding sites, zinc:copper ratio can reflect the availability of zinc in the body. The computation of this ratio revealed that the ratio was significantly lower in the patients than those in the controls during the luteal phase. Conclusions: Our data suggest that zinc deficiency occurs in PMS patients during the luteal phase, and the availability of zinc in PMS patients during the luteal phase is further reduced by the elevated copper. Fertil Steril 1994;62: Key Words: Premenstrual syndrome, zinc, copper Many theories have been proposed to understand the pathophysiology of premenstrual syndrome (PMS) in the hope of finding an effective treatment (1). The Biskinds (2) were the first to propose the role of nutritional factors in PMS. Over the last few years, nutritional supplements have been widely used as a treatment for PMS (3,4). The use of these supplements is based on the assumption that PMS Received September 1, 1993; revised and accepted March 31, * Presented in part at the 46th Annual Meeting of The American Fertility Society, Washington, D.C., October 13 to 18, 1990, and the 72nd Annual Meeting of The Endocrine Society, Atlanta, Georgia, June 20 to 23, t Reprint requests: C. James Chuong, M.D., M.P.H., Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas (FAX: ). :j: Department of Obstetrics and Gynecology, University of Texas Medical Branch. patients consume more refined sugar, refined carbohydrates, and dairy products, and less minerals, such as zinc than do normal women (3). A report on the successful treatment of PMS using megadoses of vitamins and minerals, including zinc, has appeared in the literature (3). However, in that study only two of the four subgroups classified by the predominant symptoms responded to the treatment. Zinc and vitamin A supplements were found to be effective in controlling premenstrual flare-up of oily skin and acne (5), but there are no reports on single-mineral therapy, such as zinc in the treatment of various premenstrual symptoms, and zinc deficiency was never demonstrated in PMS patients. Serum zinc levels may assist in assessing a zinc deficiency in PMS patients and serve as a guideline before and during zinc treatment. Therefore, the objective of this study is to determine whether changes in peripheral zinc levels are associated with PMS symptoms. Chuong and Dawson Zinc and copper in PMS 313

2 r Because copper competes with zinc for intestinal absorption and serum protein binding sites (6), zinc:copper ratio can reflect the availability of zinc in the body. Therefore, copper levels were also measured for the computation of zinc:copper ratio. MATERIALS AND METHODS The study was approved by the Institutional Review Board. Fourteen patients, 24 to 39 years of age, with regular menses for at least six previous cycles responded to the advertisement for the study. All were interviewed by the same investigator (C.J.C.) for detailed medical history. All were from a whitemiddle class population in Southeast Texas. Twenty-four-hour dietary histories did not reveal evidence of extraordinary food intake. They had normal physical examinations by either the investigator or the referral physicians within 6 months before the study began. Thirteen patients were in general good health, and no significant medical conditions from medical history or physical examination were noted. One was excluded because of hypothyroidism. None had a history of psychiatric disorders, determined by the Schedule for Affective Disorders and Schizophrenia-Lifetime interview (7). The patients were instructed to start charting a daily diary on the 1st day of the menses throughout the entire menstrual cycle and to qualitatively measure their premenstrual complaints. The daily diary was designed by us to follow the diagnostic guidelines established by the National Institute of Mental Health (8). Each patient documented, within the last half of the cycle, at least 3 of the 18 symptoms, with at least one psychological and one somatic symptom for at least one preceding menstrual cycle. The 18 symptoms included irritability; tension or anxiety; mood swings; emotional liability; restlessness; decreased concentration; depression; aggression; poor coordination; craving for sweet or salty food; lethargy; generalized swelling; breast tenderness; abdominal bloating; swelling of the face, hands or feet; weight gain; headache; and change in bowel habits. Patients were eliminated from the study if their symptoms were present in the first half of the cycle or if they persisted for more than 2 days after the onset of menses. Also excluded were pregnant patients, patients who had been taking drugs or medications, including oral contraceptives, thyroid preparations, antithyroid medications, or any forms of vitamin, mineral, or other nutritional supplements during the 8 weeks before the study. Each subject was also instructed to complete the Menstrual Distress Questionnaire (9) on days 7 and 25 of the menstrual cycle for one cycle. They were also instructed to complete the BBT chart during the same cycle. Eleven patients returned the questionnaire and BBT chart after 1 month. All of them demonstrated a biphasic pattern with evidence of ovulation and adequate luteal phase. The Menstrual Distress Questionnaire has been described in previous studies (9). Women whose scores on the Menstrual Distress Questionnaire were >80 on day 7 or <95 on day 25 of the menstrual cycle were excluded from the study. One patient was excluded based on these criteria. Using these entry criteria, 10 patients were included in the study. The Menstrual Distress Questionnaire score was 55.2 ± 2.3 (mean ± SE) on day 7 and ± 8.2 on day 25. Thirteen female volunteers from among our institution's employees responded to the advertisement as the control group. They underwent the similar interview on medical and diet histories, and physical examination process to those for the patient group. They were all in general good health and had regular predictable menses. None of them had premenstrual symptoms or severe dysmenorrhea. Control subjects were excluded if they had been taking any drugs or medication, including vitamin or nutritional supplements during the 8 weeks before the study. They were also asked to keep a daily diary to complete the Menstrual Distress Questionnaire and the BBT chart in the same fashion as the patient group. One subject became pregnant before completing the questionnaire, and one subject did not return the questionnaire. As a result, 11 subjects were considered for the study. None of them noted significant premenstrual symptoms on the daily diary. All but one demonstrated a biphasic pattern and adequate luteal phase on the BBT chart. None of the remaining 10 subjects were excluded based on the criteria on the Menstrual Distress Questionnaire (9). The Menstrual Distress Questionnaire score before the study was 53.1 ± 2.6 (mean ± SE) on day 7 and 51.5 ± LIon day 25.. Informed consent was obtained from all of the participants. They were instructed to complete the BBT chart daily and the Menstrual Distress Questionnaire on days 7 and 25 of each menstrual cycle during the 3-month study period. After an overnight fast, the blood samples for the serum measurements of zinc, copper, LH, ~nd FSH assays were drawn between 8 and 10 A.M. Before the blood was collected, the women sat quietly for 30 minutes. 314 Chuong and Dawson Zinc and copper in PMS Fertility and Sterility

3 They gave blood at 2- or 3-day intervals, starting on day 1 of the menstrual cycle through three cycles. Ten milliliters of blood was collected in Becton Dickinson Vacutainer tubes (Becton-Dickinson Vacutainer tube WP-6526; Becton Dickinson and Co., Rutherford, NJ) that were manufactured free of contaminating trace metals and were specifically designed for trace metal studies (10, 11). The efficacy of this tube was confirmed by assessing zinc and copper in 10 randomly selected tubes from a supply box of 1,000. This assessment was accomplished by withdrawing 6 ml reagent grade water from an acid-washed (with 5% nitric acid, rinsed with reagent grade water, air dried) 100-mL beaker into each of the Vacutainer tubes. The tubes were inverted three times and incubated at room temperature (RT) for 1 hour. Subsequent analysis for zinc and copper in the water within each of the 10 V acutainer tubes showed no detectable levels of either zinc or copper. The samples were permitted to clot and centrifuged at 1,500 rpm for 7 minutes at 4 C. The supernatant serum was transferred by an acid-washed (5% nitric acid, rinsed with reagent grade water, and air dried) Pasteur pipette into polypropylene tubes (similarly washed) and stored frozen at -20 C until analyzed. The zinc and copper were measured by atomic absorption spectrophotometry (Perkin-Elmer model 303; Perkin-Elmer Corp., Norwalk, CT) with a flame less graphite furnace (Perkin-Elmer model HGA-2000) and equipped with a 1-m V strip recorder (Perkin-Elmer model RlOOA). At the time of analysis, the frozen samples were thawed at RT and suitable aliquots were diluted 1:5 in reagent grade water with gentle mixing. The zinc concentrations of the serum samples were determined by comparison with a linear curve of increasing standard concentrations (50, 70, 90, 110, 130 JLgjdL) that were prepared from a commercial certified stock solution (Fisher SZ-13, lot no ; Fisher Products, Inc., Pittsburgh, PA). The components of the graphite furnace were set as follows: [1] drying at 100 C for 20 seconds; [2] ashing at 600 C for 60 seconds; and [3] atomization at 1,800 C for 7 seconds. The detection wavelength was 215 JLm, with 10-fold scale expansion and twofold noise suppression. The accuracy of the measurements was confirmed by recovery studies of a 1:1 solution of known serum pool and 50 JLg zinc standard dl which resulted in 98% ± 2% (mean ± SE) recovery for a population of 25 measurements. The reproducibility of each measurement was 99% ± 1 % of the same population. The copper concentrations of the 1:5 serum dilution were determined by comparison with a linear curve of increasing standard solutions (50, 70, 90, 110, 130 JLgjdL) that were prepared from a commercial certified standard (Fisher Products, Inc.). The controls of the graphite furnace were set as follows: [1] drying at 100 C for 30 seconds; [2] ashing at 1,000 C for 40 seconds; and [3] atomization at 2,300 C for 10 seconds. The detection wavelength was 325 JLm, with ten-fold scale expansion and twofold noise suppression. The accuracy of the copper measurements was confirmed by recovery studies of a 1:1 solution of known serum pool and 50 JLg copper standardjdl, which resulted in 98% ± 2% recovery for a population of 25 measurements. The reproducibility of each measurement was 99% ± 1 % of the same population. The results of the metal measurements were expressed as JLgjdL. Luteinizing hormone and FSH were measured by double-antibody RIA. All reagents except the standards were obtained from the National Pituitary Agency. Luteinizing hormone and FSH standards were obtained from the World Health Organization. The intra-assay coefficients of variation (CVs) were 7.3% for LH and 6.8% for FSH, whereas the interassay CVs were 12.3% and 15% for LH and FSH, respectively. All samples from the same patient were run in duplicate in the same assay for zinc, copper, LH, and FSH. The day of LH surge was considered as the midpoint of the cycle and was designated day O. All other measurements were expressed as the number of days before or after the LH surge. The zinc and copper measurement during the follicular phase (day LH -14 to -3) of each month for each subject was the mean of all data during the follicular phases of each month. The mean of the 3 months' values for each subject was then obtained. The zinc and copper measurement during the luteal phase (day LH +3 to + 14) was calculated in a similar fashion. The paired-sample t-tests were used to test for significant intragroup changes of zinc and copper levels between the follicular and lu_teal phases. Comparisons between the control and PMS groups during the follicular and luteal phases were made by two-sample t-tests. The differences in the Menstrual Distress Questionnaire scores were also determined by the paired-sample or two-sample t-tests. The level of significance was P < Power calculations were based on a = 0.05 (twosided), {3 = 0.2 (power 80%), and a difference in zinc and copper levels of 10 JLgjdL. This yielded a recommended sample size of 8.42 or 9 in each group. Chuong and Dawson Zinc and copper in PMS 315

4 Given the same conditions, for fj = 0.1 (power 90%), the recommended sample size would be or 12. RESULTS There were no differences in age, body weight, marital status, reproductive history, menstrual history, or degree of dysmenorrhea between the patient and control groups (Table 1). The mean duration of PMS symptoms in the patient group was 60.5 months. To participate in the study, the Menstrual Distress Questionnaire score on day 7 had to be <80. The Menstrual Distress Questionnaire score on day 7 before the study for the PMS groups was not significantly different from that of control subjects (55.2 ± 2.3 versus 53.1 ± 2.6, mean ± SE). The large difference in Menstrual Distress Questionnaire score on day 25 (125.7 ± 8.2 for PMS groups and 51.5 ± 1.1 for control group) was expected, based on the criteria for inclusion in this study. A similar pattern of score changes was noted in each PMS and control subject each month during the study period. All the participants demonstrated a biphasic BBT pattern each month, suggesting ovulation and adequate luteal phase. The LH and FSH are shown in their characteristic patterns, with the day of the LH peak designated day LH o. The LH peak reached 92.3 ± 12.5 miu /ml (mean ± SE), whereas FSH was 24.2 ± 5.3 miu /ml on day LH O. In the controls, zinc values were ± 7.8p,g/ dl (mean ± SE) during the luteal phase and ± 7.1p,g/dL during the follicular phase. No significant changes were noted between the two values. In the patients, the values were 90.3 ± 4.1p,g/dL during the luteal phase, which was significantly lower than ± 3.6 p,g/dl during the follicular phase (P < 0.05). No significant changes were noted between the controls and the patients during follicular phase. However, the values were significantly lower in the patients than those in the controls during the luteal phase (90.3 ± 4.1 versus ± 7.8 p,g/dl) (P < 0.05) (Fig. 1). In the controls, copper values were 96.2 ± 2.1p,g/ dl (mean ± SE) during the luteal phase and 98.4 ± 3.2 p,g/dl during the follicular phase. No significant changes were noted between the two values. In the patients, the values were ± 3.3p,g/dL during the luteal phase, which was not significantly different from ± 4.5 p,g/dl during the follicular phase. No significant changes were noted be- Table 1 Age, Body Weight, Marital Status, and Reproductive and Menstrual Histories of PMS Patients and Control Subjects Control PMS (n = 10) (n = 10) Age (yr) Median Range 23 to to 39 Body weight Average (% of IBWt) Range (% of IBWt) 84 to to 126 Number of obese subjects:j: 1 (124% of IBWt) 2 (120% and 126% of IBWt) Marital status Single 5 3 Married 5 5 Divorced 0 2 Reproductive history Nulliparous 6 5 Parous 4 5 Menstrual history, median Menarch (y) Interval (d) Length (d) Dysmenorrhea None 2 1 Mild 4 3 Moderate 3 4 Severe 1 2 * From Chuong CJ, Dawson ER. Vitamin A levels in premenstrual syndrome. Fertil Steril 1990;54: Reproduced with permission of the publisher, The American Fertility Society. t IBW, Ideal body weight, is according to the table of the Metropolitan Life Insurance Company, :j: Obesity is defined as body weight 20% or greater above IBW. 316 Chuong and Dawson Zinc and copper in PMS Fertility and Sterility

5 Table 2 Zinc:Copper Ratio Over Three Menstrual Cycles Control PMS Subject Follicular Luteal Subject Follicular Luteal Mean ± SE 1.11 ± ± 0.12* ± 0.09t ± 0.05*t * P < t P < tween the controls and the patients during follicular phase. However, the values were significantly higher in the patients than those in the controls during the luteal phase (106.3 ± 3.3 versus 96.2 ± 2.1 p,g/dl) (P < 0.05) (Fig. 2). The computation of zinc:copper ratio for each sample revealed that the ratio was significantly lower in the patients than those in the controls during the luteal phase (0.9 ± 0.05 versus 1.18 ± 0.12) (P < 0.025). In the patients, the ratio was also significantly lower during the luteal phase than during the follicular phase (0.9 ± 0.05 versus 1.08 ± 0.09) (P < 0.05) (Table 2). Summary of zinc levels, copper levels, and zinc:copper ratio is listed in Table 3. DISCUSSION There are several mechanisms whereby zinc might have an effect on the hormonal and neurotransmitter secretion that is related to the mental and emotional state of women. For example, zinc ions inhibit opiate receptor binding in the rat brain (12) and might be an important modulator of opiate receptor binding in the central nervous system (CNS). Neurotransmitter changes in the CNS have been linked to PMS by symptom similarity and association with other emotional symptoms (1). Zinc, at both physiological and supraphysiological concentrations, can change PRL, growth hormone, and LH synthesis or secretion in vitro (13). This may be related to the findings that zinc antagonizes many calcium-mediated processes (6) and calmodulin-dependent functions (14), and PRL secretion from the pituitary gland is dependent partly on the coordinated activity of calcium. Zinc is also important for PGE 1 synthesis because it is involved in the re- lease of dihomogammalinolenic acid from its storage site in the form of a lipid ester, which is then converted to PGE 1 (6). The effect of menstrual cycle phases on zinc levels was assessed in normally menstruating women (15). Other studies also suggest that the zinc content of the human endometrium undergoes cyclic variations (16). The mechanism responsible for these changes in metal content appears to be hormone related. It has been shown that the P binding activity of the endometrium is affected by the presence of zinc in a protein-mediated process (16). Zinc deficiency and copper excess were found in patients with PMS in our study. Because copper competes with zinc for intestinal absorption and Table 3 Summary of Zinc Levels, Copper Levels, and Zinc:Copper Ratio Follicular Luteal Zinc (Ilg/ dl) PMS ± 3.6* 90.3 ± 4.1*t Control ± ± 7.8t Copper (llg/dl) PMS ± ± 3.3+ Control 98.4 ± ± 2.1+ Zinc:copper PMS 1.08 ± ± Control 1.11 ± ± * Values are means ± SE. Comparison between zinc levels during the follicular phase and during the luteal phase in PMS patients. P < t Comparison of zinc levels during the luteal phase between the PMS patients and the controls. P < Comparison of copper levels during the luteal phase between the PMS patients and the controls. P < Comparison between zinc:copper ratio during the follicular phase and during the luteal phase in PMS patients. P < II Comparison between zinc:copper ratio during the luteal phase between the PMS patients and the controls. P < Chuong and Dawson Zinc and copper in PMS 317

6 *+ t 80 a- Wl "il.! ~ Follicular Menstrual cycle Luteal Figure 1 Zinc levels (mean ± SE) over three menstrual cycles. (_, control; D, patient). serum protein binding sites (6), the zinc:copper ratio can reflect the availability of zinc in the body. This ratio was significantly lower in patients during the luteal phase, suggesting that the availability of zinc in patients during the luteal phase was reduced further by the elevated copper. Serum zinc is tightly bound to globulin, loosely bound to albumin, and ionized with leukocytes (17). Serum copper is tightly bound to ceruloplasmin, one of the globulin, and loosely bound to albumin (18). Jimenez-Jimenez et al. (11) measured the levels of zinc and copper binding proteins to determine the functional levels of zinc and copper. However, none of the serum proteins were measured in our study because of the conflict between the large quantity of the blood required for the protein analysis and the number of samples taken in this study. Although the red blood cells (RBCs) contain a large quantity of zinc (predominantly in carbonic anhydrase carbonate) (19) that is frequently measured in zinc studies, there is no comparable quantity of copper measured within the RBCs. Consequently, the measurement of RBCs trace metals would not provide an equitable comparison of zinc and copper metabolism. Because only serum levels were measured, the possibility remains that the decreased levels we observed in the zinc during the luteal phase could be contributed by shifts from extracellular to intracellular sites. Altered gastrointestinal absorption during the luteal phase could also contribute to these changes. Although measurement of the levels of carrier proteins (a2 microglobulin and albumin) and trace metal levels in RBCs were thought to offer additional information on trace metal excess or deficiency, most metalloproteins are subject to considerable variation because of factors, such as 318 Chuong and Dawson Zinc and copper in PMS hemolysis, individual apoprotein production, and body fluid volume. At this time, there is still no universally accepted and reliable methods of demonstrating trace metal deficiency states. Our study was limited to the measurement of serum levels only, and the results should be interpreted as a suggestion, rather than a demonstration, of deficiencyexcess states of zinc and copper. 'The total serum zinc levels of the normal nonpregnant, nonlactating adult female ranges between 70 and 180 ~g/dl, and the total serum copper levels range between 75 and 153 ~g/dl (20, 21). These values have been confirmed in our laboratory. The ranges of measured serum levels of the subjects of this study were between 73 and 138 ~g/ dl for zinc and between 75 and 150 ~g/dl for copper, all of which were within the normal ranges. Because the decreased zinc levels were not below the normal range, the effects of subtle changes of zinc and copper during the menstrual cycle on various neurotransmitters requires further investigations. Because of the rigorous initial entry criteria, such as at least 2-month medication-free (including any forms of vitamin, mineral, or nutritional supplements) interval before testing and the requirement of blood drawing at 2- or 3-day intervals for 3 months when no treatment could be given, the sample size in our study was limited. Yet, the differences detected in the peripheral zinc and copper levels in PMS patients from the controls during the luteal phase were significant. It is suggested that the treatment of PMS has been hampered by a lack of laboratory evidence of the nutritional state of these patients. Stewart (4) evaluated the nutritional state of 11 PMS patients , * ~100 :a! 80 1 Wl 60.. ~ o Follicular Menstrual cycle Luteal Figure 2 Copper levels (mean ± SE) over three menstrual cycles. (_, control; D, patient). Fertility and Sterility

7 by taking at one time samples of blood and sweat, but not the controls. No specific days of the menstrual cycle were mentioned when the samples were taken. He found evidence of deficiencies in vitamin Bs and magnesium. Mean serum zinc and copper levels were within normal range, although sweat zinc levels were lower than normal levels. Copper levels were not measured in the sweat. Abraham (22) did not find significantly different serum or urine levels of zinc and several other nutrients between the follicular and luteal phases of the menstrual cycle in 6 PMS patients. However, only two blood samples during each phase were drawn, and no samples were obtained from the controls for comparisons. Mira et al. (23) measured several vitamins and trace elements in the serum of PMS patients and the controls on two occasions (premenstrual and postmenstrual) in 1 month and found no evidence for nutritional deficiencies including zinc. Copper levels were not measured in the latter two studies (22, 23). Deuster et al. (15) found plasma zinc concentrations were higher during menses and the follicular phase and then dropped during the ovulatory and luteal phases in five normally menstruating women who did not have PMS. However, the daily blood samples were obtained for only one menstrual cycle that ranged from 24 to 31 days in length. No ovulation prediction kit was used, and four phases of menstrual cycle were classified based on the cycle length and BBT. In contrast, we did not demonstrate significant changes in serum zinc levels between the follicular and luteal phases in the 10 control subjects who had no PMS, which was in agreement with the findings of Mira et al. (23). Our approach, using a more comprehensive schedule by obtaining blood samples at 2- or 3-day intervals through three menstrual cycles from both patient and control groups and using LH surge based on ovulation prediction kit to classify the menstrual phases, may explain the current findings different from previous observations. Despite the wide use of nutritional supplements containing a variety of minerals in the treatment of PMS, either an excess or deficiency of such minerals has not been well documented in affected patients compared with control subjects. Lower serum and intracellular magnesium levels during the luteal phase in PMS patients compared with control subjects have been reported (24). Stewart (4) also found evidence of magnesium deficiencies in RBCs and sweat, but not in the serum in PMS patients. Calcium deficiency in PMS patients has not been documented, although Penland et al. (25) reported that calcium intake decreased menstrual cycle symptoms recently. Essential minerals may play physiological roles in regulating the release and/or biological activity of hormones and neuropeptides involved in the menstrual cycle (12-14). Further clarification of the roles of zinc and copper and their relationships with magnesium, calcium, and other minerals in central neurotransmitters activities and the relationship between the central and peripheral levels of minerals is needed. Acknowledgments. We thank Edward R. Smith, Ph.D., Department of Obstetrics and Gynecology, University of Texas Medical Branch, Galveston, Texas, for excellent laboratory assistance in performing the LH and FSH assays and Ms. Kathy Brennan for excellent editorial assistance in the preparation of the manuscript. REFERENCES 1. Reid RL, Yen SSC. Premenstrual syndrome. Am J Obstet GynecoI1981;139: Biskind MS, Biskind GR, Biskind LH. Nutritional deficiency in the etiology of menorrhagia, metrorrhagia, cystic mastitis, and premenstrual tension. 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