0021-972X/99/$03.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1999 by The Endocrine Society
Vol. 84, No. 2 Printed in U.S.A.
Disappearance of Endogenous Luteinizing Hormone Is Prolonged in Postmenopausal Women* JULIE L. SHARPLESS, JEFFREY G. SUPKO, KATHRYN A. MARTIN, JANET E. HALL
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Reproductive Endocrine Unit (J.L.S., K.A.M., J.E.H.), and Oncology Division (J.G.S.), Department of Medicine, Massachusetts General Hospital, Boston, Massachusetts 02114 ABSTRACT Pituitary secretion of LH is increased after menopause, but it is not known whether changes in LH clearance also contribute to elevated serum levels. To determine whether the disappearance of endogenous LH is decreased in postmenopausal women (PMW), compared with normal cycling women, GnRH receptor blockade was used to inhibit endogenous secretion of LH and the glycoprotein free a-subunit (FAS), and the decline of serum levels was monitored. The NAL-GLU GnRH antagonist ([Ac-D-2Nal1,D-4ClPhe2, D-3Pal3,Arg5,D-4-p-methoxybenzoyl-2-aminobutyric acid6,D-Ala10]GnRH) was administered sc, at doses of 5, 15, 50, and 150 mg/kg, to 15 euthyroid PMW in 21 studies. Blood was sampled every 10 min, for 4 h before and 8 h after a single sc injection of the GnRH antagonist, followed by hourly samples, ending at 20 h after injection. Results of the maximally suppressive doses (50 and 150 mg/kg) were compared with those of 24 normal cycling women in the early follicular phase and late follicular phase or early luteal phase, and 8 women at the midcycle surge (MCS), who also received these doses of the GnRH antagonist. The best fit curve describing the decay of hormone serum
levels after maximal GnRH receptor blockade was determined by nonlinear regression analysis. The elimination of both LH and FAS, after GnRH receptor blockade, exhibited apparent first-order kinetics characterized by a single exponential phase. No differences were seen in percent suppression or half-lives (t ⁄ ) of LH or FAS, between the 50- and 150-mg/kg antagonist doses, in any of the subject populations; and percent suppression of LH was similar across all groups. The t ⁄ of LH was prolonged in PMW (139 6 35 min, mean 6 est. SD), in comparison with both the MCS (78 6 20 min; P , 0.0005) and other cycle stages (57 6 28 min; P , 0.0001). However, the disappearance of FAS was not different in PMW, compared with MCS or other cycle stages (t ⁄ 5 51 6 26, 41 6 12, and 41 6 19 min, respectively). Our conclusions were: 1) Disappearance of endogenous LH after GnRH receptor blockade is significantly prolonged in PMW, compared with the MCS or other cycle stages; 2) The disappearance of FAS is not altered in PMW, suggesting that differences in the disappearance of LH relate to LH microheterogeneity rather than systemic factors. (J Clin Endocrinol Metab 84: 688 – 694, 1999) 12
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T MENOPAUSE, ovarian production of sex steroids and inhibins ceases, and pituitary secretion of gonadotropins is released from feedback inhibition. Serum LH levels are increased in castrate animals and after natural or surgical menopause in women (1). LHb messenger RNA levels are increased in ovariectomized (compared with intact) rats (1), suggesting an increase in hormone synthesis. In postmenopausal women (PMW), there is evidence of an increase in LH production rates (2) and increased LH responsiveness to GnRH (3). Considerably less is known about potential changes in the clearance of LH after the menopause. Studies in which LH clearance was assessed by a single bolus or constant infusion of 131I-labeled human LH indicate that clearance of exogenous hormone is not affected by menopausal status, but they do not address the question of whether there are changes in the clearance of endogenously secreted hormone after menopause (2, 4). It is known that multiple isoforms of LH, differing in their carbohydrate structure and charge, coexist in both pituitary and serum (5, 6). Further evidence suggests
that biologic and immunologic activity, as well as hormone clearance, may vary in relation to changes in the carbohydrate structure of LH (7, 8), as has been demonstrated for FSH (9). For both LH and FSH, more basic isoforms are associated with shorter half-lives (t ⁄ ). A greater preponderance of the less basic forms is seen in PMW, compared with normal cycling women (5), suggesting that the t ⁄ of endogenous LH may be increased after menopause. This hypothesis is supported by studies showing that clearance of LH after hypophysectomy is prolonged in ovariectomized rats, compared with intact rats (10). Although the t ⁄ of endogenous LH has also been assessed in castrate women after hypophysectomy (2, 11), similar comparative data are not available in women with regular menstrual cycles. Modeling of LH clearance, using deconvolutional analysis of pulsatile data, suggests that the disappearance of endogenous LH is prolonged in PMW, compared with younger women (12). An alternative approach is to examine the disappearance of LH after GnRH receptor blockade, which inhibits endogenous LH secretion and permits a more direct assessment of hormone elimination. Thus, to test the hypothesis that the plasma disappearance of endogenous LH is decreased after menopause, GnRH receptor blockade was instituted, using maximally suppressive doses of the NAL-GLU GnRH antagonist ([Ac-D2Nal 1,D-4ClPhe 2,D-3Pal 3,Arg 5,D-4-p-methoxybenzoyl-2aminobutyric acid6,D-Ala10]GnRH), and the decline of serum LH was measured in PMW and normal cycling 12
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Received July 15, 1998. Revision received September 15, 1998. Rerevision received October 16, 1998. Accepted October 16, 1998. Address all correspondence and requests for reprints to: Dr. Janet Hall, Reproductive Endocrine Unit, Massachusetts General Hospital, Boston, Massachusetts 02114. * This work was supported by Grants R-01-AG-13241, P-30-HD28138, and M-01-RR-01066. Dr. Sharpless was supported by Grant T-32-DK-07028 –22.
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women. The decline in glycoprotein free a-subunit (FAS) was also assessed to investigate possible age-related changes in clearance mechanisms. Subjects and Methods Subjects PMW. Twenty-one studies were performed in 15 healthy, euthyroid women, 49 – 68 yr old, all of whom were at least 2 yr post menopause. Four women had undergone surgical menopause. All subjects had been off any estrogen replacement for at least 6 months before the studies. The study was approved by the Subcommittee on Human Studies of the Massachusetts General Hospital, and all participants provided written informed consent. Normal cycling women. Studies in PMW were compared with previously published results in 50 regularly cycling women, 18 – 40 yr old, who were studied in the early follicular phase (EFP), late follicular phase (LFP), and early luteal phases (ELP), (EFP1LFP1ELP combined as normally cycling women) and at the gonadotropin midcycle surge (MCS) (13). Women at the MCS were included, because their mean LH levels are comparable with those in PMW. All controls had normal TSH and PRL levels and had ovulated in the cycle before study, as evidenced by a midluteal-phase plasma progesterone level more than 6 ng/mL (.19 nmol/L) or a biphasic body temperature chart.
Experimental protocol To determine the acute response to GnRH receptor blockade, the subjects were admitted to the General Clinical Research Center of the Massachusetts General Hospital. For each study, blood was sampled every 10 min, for 4 h before and 8 h after sc injection of 5, 15, 50, or 150 mg/kg of the NAL-GLU GnRH antagonist, followed by hourly samples, ending 20 h after the antagonist administration. Five to six studies were performed at each antagonist dose. For those women who participated in more than one study, studies were performed at least 30 days apart, to ensure recovery from any effect of the antagonist or blood drawing, and no subject was studied at the same dose on more than one occasion. A total of 63 studies were performed in the control subjects in the EFP, LFP, and ELP; with 12, 23, 14, and 14 normal subjects studied at the 5-, 15-, 50-, and 150-mg/kg doses, respectively. Sixteen subjects who were studied at the MCS were compared separately.
Assays Serum concentrations of LH, FSH, and FAS were measured by RIA, using a b-directed LH polyclonal RIA, an intact-directed FSH polyclonal RIA, and a monoclonal RIA specific for FAS, as previously described (14, 15). All samples from an individual subject’s study were measured in duplicate in the same assay. The intraassay coefficients of variation for LH and FSH were between 4 and 7%; the interassay coefficients of variation for LH and FSH were between 3 and 6%. The LH and FSH assays had a sensitivity of 0.8 IU/L using the Second International Reference Preparation of human menopausal gonadotropin as the standard. For FAS, the intraassay coefficient of variation was 3–9%, and the interassay coefficient of variation was 4 –12%. The sensitivity of the FAS assay was 30 ng/L. Estradiol (E2) was measured by RIA after extraction, as previously described (16). The sensitivity of the assay was 20 pg/mL.
Data analysis To determine the extent of the LH, FAS, and FSH decrease after administration of the GnRH antagonist in PMW, the data for each patient were divided into a pretreatment 4-h baseline period and five 4-h periods after antagonist administration. Mean hormone levels from each time period were then compared, using ANOVA for repeated measures, followed by post hoc Newman-Keuls testing for individual differences. The percent inhibition of each hormone was calculated for each dose, as previously described (13), by determining the nadir after antagonist administration, subtracting this from the mean baseline value, and expressing the difference as a percent of baseline. The nadir was determined for LH and FAS (using a 6-point moving average) and for FSH
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(using a 3-point moving average). There was no difference in the percent inhibition among the EFP, LFP, and ELP (13); therefore, the data from each dose during the different stages were combined and used for reference. The maximum percent inhibition was then compared among the three glycoprotein hormones and among PMW, the normal cycling women, and women at the MCS, using ANOVA. Kinetic parameters describing the decrease of gonadotropin serum concentration after the administration of maximally inhibitory doses of the GnRH antagonist to normal cycling and PMW were determined by nonlinear least-squares regression of the raw data using the WinNonlin Version 1.1 software package (Scientific Consulting, Apex, NC). The empirical equation that best fit the observed time courses was established by visual inspection of the predicted profile, residual analysis, the sum of squared residuals, the degree of correlation between model parameters, and the magnitude of the coefficients of variation of the parameter estimates (17). Only subjects studied at doses of 50 or 150 mg/kg who demonstrated full GnRH receptor blockade (defined by the absence of pulsatile secretion, as assessed by CLUSTER 232 analysis) were included in the final analysis. There was no difference in t ⁄ of LH or FAS among normal cycling women in the EFP, LFP, and ELP; and these cycle stages were combined. Thus, the characteristics of LH disappearance were analyzed in 10 PMW, 24 normal cycling women, and 8 MCS studies. FAS disappearance rates were analyzed in 9 PMW, 10 normal cycling women, and 6 MCS subjects; and the results were compared with those of LH, using unpaired t tests. Because FAS results were available in fewer subjects than LH, a matched comparison was also performed using only subjects in whom both were available. Hormone levels and percent inhibition are expressed as the mean 6 sem unless otherwise specified. Mean values of the apparent gonadotropin t ⁄ for each group of subjects were calculated as harmonic means and are reported together with the jackknife estimate of the standard deviation (18). A P value , 0.05 was considered to be significantly different. 12
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Results Baseline characteristics
At baseline, the 15 PMW were an average of 12.3 (range, 2–22) yr from menopause, with an LH of 66.1 6 4.3 IU/L (mean 6 sem); FSH, 127.4 6 10.7 IU/L; and FAS, 874.3 6 86.1 ng/L. E2 was at or below the limit of detection (20 pg/mL) in all subjects, with the exception of 1 woman studied 7 yr after her last episode of menses, whose E2 level was 33 pg/mL. At baseline, LH, FSH, FAS, or E2 did not differ among dose groups. Inhibition of LH, FSH, and FAS by GnRH receptor blockade in PMW
GnRH receptor blockade in PMW resulted in a decrease in LH in response to all doses of the NAL-GLU GnRH antagonist (P , 0.0001; Fig. 1). At each dose, LH levels remained significantly lower than baseline, for up to 20 h post antagonist (P , 0.0002). However, at the two lower doses, some recovery occurred within the 20-h sampling window. Pulsatile secretion of LH was abolished in all subjects at the 50and 150-mg/kg antagonist doses. FSH similarly decreased after all doses of GnRH antagonist administered (P , 0.0002; Fig. 1), without evidence of significant recovery during the 20 h of observation after antagonist administration at any dose. FAS was suppressed at all doses of the antagonist (P , 0.03 for doses of 5 and 15 mg/kg; P , 0.005 for 50 and 150 mg/kg; Fig. 1). At the highest two doses, FAS remained fully suppressed for the duration of the study. Only one postmenopausal subject demonstrated continued pulsatile secretion of FAS after the two highest doses of the NAL-GLU antagonist
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FIG. 1. Mean (6 SEM) serum LH, FSH, and FAS in PMW at baseline and after administration of the NAL-GLU GnRH antagonist at the doses indicated. The timing of antagonist administration is indicated by the dotted line.
(and was therefore excluded from analysis of hormone disappearance).
between PMW and normal cycling women (40.6 6 1.6 and 43.6 6 2.8%), whereas percent inhibition was greater at the MCS (53.4 6 4.8%; P , 0.05), as previously described (13).
Maximum percent inhibition
The maximum percent inhibition of LH after GnRH antagonist administration in the PMW (Fig. 2) was similar between doses of 5 and 15 mg/kg (58.9 6 2.7 and 59.5 6 3.5%, respectively) and significantly less than that seen with a dose of 50 mg/kg (75.2 6 3.4%; P , 0.01). There was no further suppression at the 150-mg/kg dose (78.6 6 1.5%). The maximum percent inhibition of LH at the 150-mg/kg dose was not different between PMW and normal cycling women (80.7 6 1.7%) but was somewhat greater at the MCS (84.3 6 1.3; P , 0.05 vs. PMW). The maximum percent inhibition of FAS was similar between doses of 50 and 150 mg/kg. The maximum inhibition at the highest antagonist dose was significantly greater in PMW than in normal cycling women (49.2 6 2.4 vs. 29.3 6 2.7%; P , 0.001) but not different from the MCS (51.5 6 4.1%). The maximum percent inhibition of FSH occurred at an antagonist dose of 50 mg/kg, with no further suppression at the 150-mg/kg dose in PMW. There was no difference in maximum percent inhibition of FSH
Kinetics of endogenous LH and FAS disappearance from serum
Expression of the data as percent of baseline revealed the existence of a marked difference in the pattern of hormone disappearance after GnRH receptor blockade, between PMW and normal cycling women, for LH (Fig. 3) but not for FAS. Serum concentration-time courses of LH and FAS, in subjects who received 50 and 150 mg/kg of the NAL-GLU GnRH antagonist, were analyzed to characterize their elimination kinetics. After antagonist administration, the rapid decline of serum LH and FAS concentrations (C) to a constant nadir level (Cmin) was best described in all subjects by an equation with a single exponential term:
C 5 C9e2k,t 1 Cmin where t is time after dosing, C9 is the difference between the initial serum hormone concentration (C0) and Cmin, and ke is
LH DISAPPEARANCE IN POSTMENOPAUSAL WOMEN
FIG. 2. Percent inhibition of LH and FAS in response to increasing doses of the NAL-GLU GnRH antagonist in PMW (F), normal cycling women (E), and women at the MCS (D) (mean 6 SEM). Maximum percent inhibition of LH was not different between groups. In PMW, percent inhibition of FAS at the highest antagonist dose was greater than in normal cycling women but not different from women at the MCS.
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FIG. 4. Representative example of the fit of the nonlinear regression model used to estimate serum t ⁄ of LH and FAS. The NAL-GLU GnRH antagonist was administered at 4 h. Hormonal data is presented on a log scale, as per pharmacokinetic convention. 12
recover, and the drug concentration was not measured. Because FSH continued to decline and did not approach a nadir during the course of this study, a t ⁄ was not modeled. Harmonic mean t ⁄ for the elimination of LH and FAS by each cohort of subjects studied, calculated from nonlinear regression estimates of the ke values for individual subjects, are summarized in Table 1. In PMW, the t ⁄ of LH was 139.1 6 35.4 min and was significantly prolonged, in comparison with both the normal cycle (56.9 6 28.4 min; P , 0.0001) and the MCS (77.5 6 20.1 min; P , 0.0005). The LH t ⁄ at the MCS was also significantly longer than at the other cycle stages (P , 0.01). The t ⁄ of FAS was shorter than that of LH in all three groups (P , 0.05; Table 1). In contrast to LH, there was no difference in the t ⁄ of FAS among PMW, normal women, and women at the MCS (50.5 6 25.8, 41.2 6 12.0, and 40.7 6 19.1 min, respectively). All statistical differences remained significant in the matched comparison using only subjects with both LH and FAS data. 12
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FIG. 3. LH, expressed as a percent of the mean baseline value, in PMW (F) and normal cycling women (–) (mean 6 1 SEM). The timing of the NAL-GLU GnRH antagonist administration is indicated by the dotted line.
Discussion
Using GnRH receptor blockade as a physiologic probe to assess the pharmacodynamics of endogenous LH, we have shown that the t ⁄ of LH in PMW is 2.5 times that found in women with regular ovulatory cycles and twice that seen in women at the midcycle surge (MCS). Assessment of the disappearance of FAS after GnRH receptor blockade served as an important control in these studies. The t ⁄ of that portion of FAS that is controlled by GnRH was not altered in PMW, and thus, differences in plasma disappearance of LH are 12
the first-order rate constant for serum hormone decay. The excellent characterization of the observed data by this model, for both LH and FAS, is demonstrated in Fig. 4. Development of a more highly descriptive pharmacodynamic model was precluded because sampling was discontinued at 20 h after dosing, before serum LH and FAS concentrations began to
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TABLE 1. Mean gonadotropin levels and half-lives LH Baseline (IU/L) Mean 6 SEM
Postmenopausal women Normal cycling women EFP 1 LFP 1 ELP MCS
FAS t1/2 (min) Mean 6 est SD
Baseline (ng/L) Mean 6 SEM
t1/2 (min) Mean 6 est SD
62 6 3a
139 6 35a,b
774 6 45a
51 6 26
10 6 1 56 6 11a
57 6 28 78 6 20c
266 6 44 627 6 122d
41 6 12 41 6 19
P , .0001 vs. EFP1LFP1ELP. P , .0001 vs. MCS. c P , .01 vs. EFP1LFP1ELP. d P , .005 vs. MCS. a b
unlikely to result from changes in renal or hepatic clearance secondary to aging or hormonal state. The prolonged disappearance of LH in PMW is most likely caused by differences in the isoform composition of secreted LH. Exogenous injection of different LH isoforms in animal studies has shown that the metabolic clearance rate varies with isoform (19). In general, animal studies have shown that more basic forms of both LH and FSH yield a greater in vitro potency, but shorter t ⁄ in circulation, whereas more acidic isoforms have a longer circulatory time and are more active in in vivo estimations (7, 20). Changes in isoform composition, to more acidic forms, may be caused by decreased activity of the GalNAc-transferase, with subsequent increases in LH sialic acid content (21). The longer t ⁄ of LH in PMW in the current studies is consistent with the presence of a preponderance of the more acidic forms of LH, which have been described in PMW (5, 22). In the current studies, we have defined the dose-response relationship for GnRH receptor blockade, using the NALGLU GnRH antagonist in PMW, and we demonstrated that a dose of 50 mg/kg causes maximum suppression, with no further change at 150 mg/kg. At these doses, pulsatile secretion of LH was abolished for up to 20 h; and serum LH values declined, to a constant nadir, permitting evaluation of the elimination rate constant. The maximum degree of LH suppression in these studies is similar to that reported previously for both immunoactive (23–25) and bioactive (26) LH in PMW and normal cycling women (13, 27) using a variety of potent GnRH antagonists. Although it is known that secretion of FAS is under the dual control of GnRH and TRH, the current studies, in which FAS pulses were abolished by the highest doses of the GnRH antagonist, provide additional support for the hypothesis that the pulsatile component of FAS secretion is predominantly controlled by GnRH (28, 29). Maximum inhibition of FAS was greater in both PMW and at the MCS, in comparison with normal cycling women. These results suggest that, in these situations, in which the pituitary response of LH to GnRH is enhanced (3, 30), GnRH also contributes a greater proportion to overall FAS secretion, whereas the thyrotrope contribution of FAS remains fixed. The use of GnRH receptor blockade is analogous to the limited number of studies in which the disappearance of endogenous LH was calculated after hypophysectomy (11), but it has the advantage of permitting comparison with normal women. Previous studies, which have used deconvolution techniques on individual pulses, have estimated the LH t ⁄ as 150 –171 min in PMW (slightly longer, but generally 12
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consistent with the current studies) (12, 31). Estimation of t ⁄ , using blockade of LH secretion, offers the advantage (over deconvolution techniques) of more direct measurement and increased accuracy, because of an increased number of samples in the decline and the ability to follow the decline over several half-lives. At the maximally inhibitory doses of the NAL-GLU antagonist, a small amount of residual LH persists. Residual LH may result from non-GnRH-dependent secretion, forms of LH with an extremely long clearance, or lack of complete blockade of the GnRH receptor by this antagonist. If the GnRH receptor is not completely blocked by the GnRH antagonist, ongoing secretion could potentially influence the calculated t ⁄ of LH. However, because the rate of decline of LH is so rapid after GnRH antagonist administration, the rate constant of formation would be insignificant, relative to that of elimination. In addition, both the maximum percent inhibition and the absolute residual LH concentration were similar in the postmenopausal and MCS subjects; and thus, this mechanism could not account for the prolonged t ⁄ of LH in PMW, compared with the MCS. In the current studies, the disappearance of LH after GnRH receptor blockade was markedly prolonged in PMW, compared both with normal cycling women and with women at the MCS. Previous studies have suggested that clearance mechanisms are saturable at high plasma hormone concentrations (32). Thus, women studied at the MCS served as an important control group, because mean LH was similar to that in PMW, suggesting that the observed decrease in clearance in PMW does not result from saturation of clearance mechanisms. The t ⁄ of FAS is significantly shorter than that of LH in both PMW and normally cycling women, consistent with studies in which clearance of LH and FAS was estimated from endogenous pulsatile secretion in GnRH-deficient men receiving exogenous GnRH (33). We have only assessed that portion of FAS that is presumed to be derived from the gonadotrope in these studies. Importantly, the estimated t ⁄ of FAS was similar between studies in which TSH-a or LH-a were infused into normal subjects (4, 34), suggesting that the cellular source of FAS may not influence its clearance characteristics. Mean FAS is elevated both in PMW and in women studied at the MCS; and thus, the disappearance of FAS, as for LH, is not influenced by mean level. Previous studies, assessing gonadotropin t ⁄ , have variously reported one and two components of clearance. Where reported, the first component t ⁄ ranges from 10 – 60 min (2, 12
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11, 20, 35) and is attributed to either serum distribution or hepatic clearance. Use of a receptor blockade model and an endogenously pulsatile environment may have masked this rapid component in the current studies. Pituitary glycoprotein hormones are cleared by both renal (36, 37) and hepatic (38, 39) mechanisms. Previous studies failed to demonstrate an effect of menopause on clearance of exogenous TSH-a (34) or an effect of gender on clearance of exogenous hCG-a (40). In men, age did not affect clearance of endogenous FAS, assessed by deconvolution (41). These results are consistent with the lack of effect of physiologic state on the disappearance of endogenous FAS in the current studies, and they imply that renal and/or hepatic handling of glycoprotein hormones is not generally impaired secondary to age or gonadal hormone status in PMW. Metabolic clearance of exogenous LH is not different in PMW (2, 4) or ovariectomized animals (10), in comparison with the gonadally intact state, supporting this conclusion and suggesting that the prolonged disappearance of endogenous LH in the current studies must be caused by changes in the isoform composition of LH secreted in PMW. In summary, the GnRH antagonist provides a unique physiologic probe that permits inferences to be made about the disappearance of the secretory products of the gonadotrope. The t ⁄ of endogenous LH, after GnRH receptor blockade, is significantly prolonged in PMW, compared with the MCS or other cycle stages, whereas the disappearance of FAS does not change. These data suggest that differences in the disappearance of LH in PMW relate to changes in LH microheterogeneity, rather than systemic factors. Further studies will be required to determine whether changes in LH disappearance in PMW are related to effects of aging or loss of gonadal feedback.
10. 11. 12.
13.
14. 15. 16. 17. 18. 19. 20.
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Acknowledgments We gratefully acknowledge the technicians of the RIA core laboratory of the Reproductive Endocrine Sciences Center for their superb technical contributions to this study; and the nurses from the General Clinical Research Center (M-01-RR-01066) for their care of the subjects. We thank Dr. Patrick Sluss for his careful review of this manuscript. The NAL-GLU GnRH antagonist was synthesized at the Salk Institute (under NIH Contract N-01-HD-0 –2906) and made available by the Contraceptive and Reproductive Health Branch, Center for Population Research, National Institute of Child Health and Human Development.
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Bone and Tooth Society Meeting June 22-24, 1999 Bristol, United Kingdom Organizers: John Beresford (Bath) and Jon Tobias (Bristol). Abstract deadline is March 1, 1999. Further details available from Janet Crompton, 29 North Road, St. Andrews, Bristol BS6 5AD, United Kingdom. Telephone: 144-117-924-8160; fax: 144-117-924-1208; E-mail:
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