Lipid, glucose and homocysteine metabolism in women treated with a GnRH agonist with or without raloxifene

Stefano Palomba1,5, Tiziana Russo1, Francesco Orio, Jr2, Annalidia Sammartino3, Francesco Maria Sbano1, Carmine Nappi3, Annamaria Colao2, Pasquale Mastrantonio4, Gaetano Lombardi2 and Fulvio Zullo1

1 Department of Obstetrics & Gynaecology, University of Catanzaro ‘Magna Graecia’, Catanzaro, 2 Department of Molecular & Clinical Endocrinology and Oncology, University of Naples ‘Federico II’, Naples, 3 Department of Gynaecology Obstetrics and Human Reproduction, University of Naples ‘Federico II’, Naples and 4 Department of Obstetrics & Gynaecology, University of Messina, Messina, Italy

5 To whom correspondence should be addressed at: Via Nicolardi 188, Napoli 80131, Italy. e-mail: stefanopalomba{at}tin.it


    Abstract
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
BACKGROUND: Although GnRH analogues are widely used to treat a variety of sex hormone-related diseases, little is known about their effect on metabolism. Therefore, we have evaluated the effect of a GnRH analogue, administered with or without raloxifene, on serum levels of lipoproteins, glucose, insulin and homocysteine (Hcy). METHODS: One hundred premenopausal women with symptomatic uterine leiomyomas were initially enrolled and randomized to receive 3.75 mg/28 days leuprolide acetate depot associated with 60 mg/day raloxifene hydrochloride (group A) or 1 placebo tablet/day (group B) for six cycles of 28 days. At entry and at cycle 6, subjects underwent anthropometric measurements, including body mass index and waist-to-hip ratio measurements, and blood chemistry assays for serum total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), triglycerides (TG), glucose, insulin, Hcy, vitamin B12 and folate concentrations. Insulin resistance was evaluated with the homeostasis model assessment (HOMA) score. RESULTS: Baseline parameters were similar in the two groups. At cycle 6, TC, HDL-C, LDL-C and TG levels were significantly increased (P < 0.05) in group B. In group A, LDL-C levels were unchanged, and TC, HDL-C and TG levels were increased (P < 0.05). Serum TC and LDL-C levels differed (P < 0.05) between the groups. Glucose levels were unchanged between and within groups, whereas insulin levels and HOMA scores increased (P < 0.05) versus baseline in group B. Post-treatment Hcy levels were higher (P < 0.05) versus baseline in group B; they were unchanged in group A. Serum vitamin B12 and folate concentrations were unchanged in both groups. CONCLUSIONS: GnRH analogues alter serum lipoprotein and Hcy levels and increase insulin resistance. These acute metabolic changes may be prevented or reduced by raloxifene.

Key words: GnRH agonist/leiomyomas/metabolism/raloxifene/selective estrogen receptor modulators


    Introduction
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Sex hormone-related conditions such as uterine leiomyomas, endometriosis and premenstrual syndrome respond well to treatment with GnRH analogues. In fact, continuous administration of GnRH analogues down-regulates pituitary GnRH receptors and, after initial stimulation of the pituitary–ovarian axis, prevents gonadotropin release and induces a state of hypogonadotropic hypogonadism (Adashi, 1994Go).

The hypoestrogenism induced by GnRH analogues frequently causes climacteric-like symptoms, i.e. vasomotor symptoms, cognitive defects and severe bone damage (Sherwin and Tulandi, 1996Go; Palomba et al., 1998Go, 2002aGo). The addition of steroid hormones to GnRH analogues (‘add-back therapy’) may reduce the deleterious effects of severe hypoestrogenism and thereby allow more prolonged treatment (Adashi, 1994Go).

Very few studies have investigated the effects of acute and severe GnRH analogue-related hypoestrogenism on lipid metabolism (Buhler et al., 1992Go; Gerhard et al., 1992Go; Cheung et al., 2000Go; Al-Omari et al., 2001Go; Somekawa et al., 2001Go). Moreover, no-one has investigated the effects of GnRH analogues with or without add-back therapy on glucose metabolism and on homocysteine (Hcy) levels, a marker of cardiovascular disease (Refsum et al., 1998Go).

In an earlier prospective randomized study, we found an altered lipoprotein pattern in women treated with GnRH analogues (Palomba et al., 1998Go). Serum levels of total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C) and triglycerides (TG) were significantly increased, whereas low-density lipoprotein cholesterol (LDL-C) levels were unchanged. Other groups reported that GnRH analogues did not affect the lipid profile (Buhler et al., 1992Go; Gerhard et al., 1992Go; Al-Omari et al., 1999Go, 2001Go; Cheung et al., 2000Go; Somekawa et al., 2001Go). More recently, Somekawa et al. (2002)Go confirmed that GnRH analogue administration alters serum lipoprotein levels and reported that ipriflavone only alleviates pharmacological hypoestrogenism.

Raloxifene hydrochloride is a synthetic non-steroidal drug derived from benzothiophene. It is a selective estrogen-receptor modulator, i.e. a compound that interacts with estrogen receptors and elicits tissue-specific responses (Khovidhunkit et al., 1999Go). Being an estrogenic agonist, raloxifene exerts a beneficial effect on metabolism, bone tissue, and the cardiovascular and central nervous systems (Walsh et al., 2001Go; Yaffe et al., 2001Go; Barrett-Connor et al., 2002Go; Cranney et al., 2002Go), without harming the breast or uterus (Cauley et al., 2001Go; Goldstein et al., 2001Go; Palomba et al., 2001Go, 2002bGo).

We found previously that the addition of raloxifene to GnRH analogues further reduces the size of the uterus and leiomyomas (Palomba et al., 2002cGo), and prevents the analogue-related bone loss in women with uterine leiomyomas (Palomba et al., 2002dGo).

The aim of the present study was to evaluate the effect of GnRH analogue administration alone and combined with raloxifene on serum levels of lipoproteins, glucose, insulin and Hcy.


    Patients and methods
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
The procedures used were in accordance with the guidelines of the Declaration of Helsinki on human experimentation. The Institutional Review Boards of the Universities of Catanzaro, Naples and Messina approved the study. The purpose of the study was explained to the patients and control women, and written consent was obtained before beginning the study.

Subjects
Between June 2000 and January 2001, 100 premenopausal women affected by symptomatic uterine leiomyomas were initially enrolled in a wider study (Palomba et al., 2002cGo, dGo). Exclusion criteria were: neoplastic, metabolic, endocrine, liver, haematological and infectious diseases; active rheumatoid arthritis; a history of acute or recurrent vascular thrombosis; bone mineral density (BMD) values <1 SD from the mean peak value for sex-matched healthy young adults (–1.0 T-score) at the posterior–anterior lumbar spine; a body mass index (BMI) of <18 or >30 kg/m2; previous or current treatment with bisphosphonates, sodium fluoride, calcitonin, estroprogestins or anabolic steroids, corticosteroids, calcium (Ca) or vitamin D, phosphate (P), thiazidic diuretics or other drugs interfering with bone metabolism; and abnormal serum levels of creatinine (Cr) and of parathyroid hormone (PTH) (Palomba et al., 2002dGo). We also excluded women smoking >20 cigarettes per day and drinking more than three alcoholic beverages per day.

Additional exclusion criteria were used for the present study. To prevent the confounding effects of hormonal and dietary factors, we excluded women with a waist-to-hip ratio (WHR) >0.8, clinical and/or biochemical hyperandrogenemia, a high folate intake (serum folate levels >12.5 nmol/l) (Orio et al., 2003Go) or hyperhomocystenaemia.

Treatment protocol
At study entry, all subjects were randomized in single blocks into a single-blind placebo-controlled study design using a computer-generated randomization list. The subjects were assigned to one of two groups of 50 women each. All women received leuprolide acetate depot (LAD) (Enantone; Takeda, Rome, Italy) at a dose of 3.75 mg/28 days in association with or raloxifene hydrochloride (Evista; Eli Lilly, Sesto Fiorentino, Italy) at a dose of 60 mg/day by mouth (group A) or placebo tablets (one tablet/day by mouth; group B). Treatment was given for six cycles of 28 days each, and single-blinding was maintained for all groups throughout the study. After cycle 6, group A women continued the treatment for a further 12 cycles.

Study protocol
At the beginning of the study and after six treatment cycles we evaluated uterine and myoma size, the number of tumours, endometrial thickness by means of transvaginal ultrasonography (Palomba et al., 2002cGo), BMD and bone turnover markers (Palomba et al., 2002dGo). We measured the weight and height of each woman to calculate the BMI. The WHR was evaluated from measurements of the waist and hip circumferences, taken at the levels of the umbilicus and upper anterior hipbone, with the subject in a standing position.

Before and after six treatment cycles, a blood sample, in duplicate determinations, was collected from each woman, immediately centrifuged, and stored at –80°C until analysis. Blood was sampled between 08.00h and 09.00h with the subject resting in bed, after overnight fasting and a 3-day 300-g carbohydrate diet. At study entry the blood samples were collected during the early follicular phase (between days 2 and 5) of the menstrual cycle. The mean of two assays was used in this study. Calcium intake, alcohol consumption and physical activity were evaluated as reported elsewhere (Palomba et al., 2002dGo).

No dietary restriction or changes were implemented during the study. All patients with a Ca intake <1000 mg received daily supplements in the form of an effervescent tablet composed of calcium carbonate (Cacit; Procter & Gamble, Rome, Italy) to ensure a total daily Ca intake of at least 1000 mg.

Each woman was asked to use barrier contraception throughout the study because of the possibility of pregnancy during the flare-up phase or in case of poor compliance to LAD treatment.

Subjects were instructed to report in the daily diary the severity leiomyoma-related symptoms, the number of uterine vaginal bleeding episodes and of hot flushes, and any adverse events.

Biochemical parameters
Serum TC, LDL, HDL, TG, glucose, insulin, Hcy, vitamin B12 and folate concentrations were evaluated in each woman at study entry and at the sixth treatment cycle. Serum TC, HDL-C, LDL-C and TG levels were measured with an autoanalyzer (MonarchTM 1000; Instrumentation Laboratory, Milan, Italy) using commercially available kits (IL TESTTM; Instrumentation Laboratory) as reported previously (Palomba et al., 1998Go, 1999Go). The reference range for serum TC was 3.63–5.70 mmol/l; the coefficient of variation (CV) (10 samples/run, 20 runs) for a sample with 4.66 mmol/l TC was 1.1% within run and 1.4% total. The reference range for serum HDL-C was 0.74–2.01 mmol/l; the CV (three samples/run, two runs/day for 10 days) for a sample with 0.89 mmol/l HDL-C was 1.9% within run and 4.0% total. The reference range for serum TG was 0.40–1.54 mmol/l; the CV (5 samples/run, 20 runs) for a sample with 1.27 mmol/l TG was 1.7% within run and 2.2% total. Serum LDL-C was evaluated using Friedewald’s formula: TC – HDL-C – 1/5 TG (Friedewald et al., 1972Go).

Plasma glucose levels were measured with the glucose oxidase method on a Beckman Glucose Analyzer (Beckman, Fullerton, CA, USA) that has a sensitivity of 0.3 mmol/l, and intra-assay and inter-assay CVs of 1.0% and 1.2%, respectively. Serum insulin was measured with a solid-phase chemiluminescent enzyme immunoassay using commercially available kits (Immunolite Diagnostic Products Co., Los Angeles, CA, USA) with a sensitivity of 2.0 µU/ml, and intra-assay and inter-assay CVs of 5.5% and 5.8%, respectively.

The homeostasis model assessment (HOMA) score, which is a measure of insulin resistance, was calculated for all subjects with the formula: fasting serum insulin (µU/ml) x fasting plasma glucose (mmol/l)/22.5 (Matthews et al., 1985Go). Serum Hcy levels were measured as previously reported (Orio et al., 2003Go) by high-performance liquid chromatography using a C-18 reverse-phase 25 x 0.46 cm column and fluorescence detection (Beckman Gold System; Beckman, Palo Alto, CA, USA), and an isocratic buffer consisting of acetate buffer 0.1 mol/l pH 4.0 (containing 20 ml methanol/l buffer). A Shimadzu RF535 fluorimetric detector (Shihadzu Corp., Kyoto, Japan) (emission {lambda} = 385 nm, excitation {lambda} = 515 nm) was used for detection. Ammonium-7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate (SBD-F) was used as a derivative agent of the Hcy thiol residue. The sensitivity of the Hcy assay was >0.25 µmol/l of serum. Acetyl-cysteine served as internal standard (acetyl-cysteine); the inter- and intra-assay CVs were 2% and 1%, respectively. Vitamin B12 and folate were analysed by capillary electrophoresis (P/ACE 5000 system; Beckman) as reported elsewhere (Orio et al., 2003Go). Separations were accomplished with a 75 µm ID silica fused capillary with a length to detector of 50 cm. The capillary was filled with a 10 mM Tris–sodium phosphate buffer pH 7.56. The applied voltage was 20 kV and detection was 200 nm. The inter- and intra-assay CVs for vitamin B12 assay were 7.4% and 6.7%, respectively, and for folate, 4.1% and 4.2%, respectively.

Statistical analysis
Because the primary end-point of our study was to determine the effect of raloxifene administration on lumbar spine BMD changes induced by GnRH analogues, our sample size was 50 subjects/group (Palomba et al., 2002dGo).

Analysis of the variance (ANOVA) for repeated measures was used to test for differences between and within the two groups in age, BMI and WHR at entry and after six cycles of treatment. Analysis of covariance (ANCOVA) was also used to compare the biochemical data between and within the two treatment groups. Wilcoxon’s signed rank test was used to compare parity, cigarettes smoked, alcohol consumption, calcium intake and physical activity. A Sperman’s rank correlation was performed to test the relationship between serum Hcy levels and vitamin B12 and folate concentrations, and between HOMA score and Hcy levels. All the parameters in the ANOVA were adjusted for chronological age, BMI, WHR, smoking, physical activity and alcohol consumption. The SPSS 11.0 (SPSS Inc., Chicago, IL, USA) package was used for statistical analyses. Continuous data were expressed as mean ± SD. A P-value <0.05 was considered statistically significant. The data were analysed using the intention-to-treat method.


    Results
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Here we report data on serum lipoprotein, glucose, insulin and Hcy levels in 42 women treated with GnRH analogues associated or not with raloxifene. Data about the effect of raloxifene addition in subjects treated with GnRH analogues on bone metabolism, uterine and leiomyoma size, and leiomyoma-related symptoms are reported elsewhere (Palomba et al., 2002cGo, dGo).

The characteristics of the patients are listed in Table I. After randomization, the two groups were similar as regards the main parameters evaluated (Table I). The number of withdrawals was similar in the two groups and no withdrawal was due to a drug-related adverse event (Palomba et al., 2002cGo, dGo). The two treatment regimens were generally well tolerated. The total incidence of all adverse events and of drug-related adverse events was similar in the two groups (Palomba et al., 2002cGo, dGo). The mean number of hot flushes per day increased significantly (P < 0.05) in both groups 15 days after starting treatment and remained constant throughout the study without between-group differences (Palomba et al., 2002cGo). There was no difference in BMI and WHR between the two groups either at baseline or after six cycles of treatment (Table I).


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Table I. Characteristics of the patients at study entry (data are expressed as mean ± SD)
 
Baseline TC, HDL-C, LDL-C and TG levels were similar in the two groups (Table I). In group B, post-treatment TC, HDL-C, LDL-C and TG levels were significantly higher than baseline values (P < 0.05) (Table I; Figure 1). In group A, post-treatment serum TC, HDL-C and TG levels were also significantly higher than baseline (P < 0.05) (Table I; Figure 1). Serum TC and LDL-C levels were significantly (P < 0.05) lower in group A than in group B (Table I; Figure 1).



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Figure 1. Percentage change in serum lipoprotein levels versus baseline after six cycles of GnRH analogue administration in association with raloxifene (group A, grey bars) or placebo (group B, open bars). *P < 0.05 versus baseline, °P < 0.05 versus group B.

 
Baseline serum fasting glucose and insulin levels and HOMA scores were similar in the two groups (Table I). After six cycles of treatment, glucose levels did not differ either between or within groups (Table I; Figure 2). Post-treatment insulin concentrations and HOMA scores were significantly higher than baseline values in group B (P < 0.05), but not in group A (Table I; Figure 2).



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Figure 2. Percentage change in serum glucose and insulin levels, and HOMA score versus baseline after six cycles of GnRH analogue administration in association with raloxifene (group A, grey bars) or placebo (group B, open bars). *P < 0.05 versus baseline.

 
Baseline Hcy, vitamin B12 and folate levels were similar in the two groups (Table I). Post-treatment Hcy levels were significantly higher than baseline values in group B (P < 0.05), and unchanged in group A (Table I; Figure 3). Serum vitamin B12 and folate concentrations remained similar between and within groups after six cycles of treatment (Table I; Figure 3).



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Figure 3. Percentage change in serum Hcy, vitamin B12 and folate levels versus baseline after six cycles of GnRH analogues administration in association with raloxifene (group A, grey bars) or placebo (group B, open bars). *P < 0.05 versus baseline.

 
At study entry, there was a significant (P < 0.05) inverse correlation between serum Hcy and vitamin B12 levels (r = –0.49 and r = –0.51 for group A and B, respectively) and folate concentrations (r = –0.47 and r = –0.49 for group A and B, respectively). Post-treatment, there was still a significant (P < 0.05) inverse correlation between serum Hcy and vitamin B12 levels (r = –0.46 and r = –0.48 for group A and B, respectively) and folate concentrations (r = –0.45 and r = –0.47 for group A and B, respectively). Similarly, there was a significant (P < 0.05) correlation between Hcy levels and HOMA score in both groups at study entry (r = +0.45 and r = +0.49 for group A and B, respectively) and after six cycles of treatment (r = +0.44 and r = +0.47 for group A and B, respectively).

Lipoprotein, glucose, insulin, Hcy and vitamin B12 levels were within normal range in all women after six cycles of treatment.


    Discussion
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Data about the effect of GnRH analogues treatment on the lipoprotein pattern and about the effectiveness of steroidal add-back treatment are scarce and not conclusive. Al-Omari et al. reported a significant increase in LDL-C and in the LDL-C/HDL-C ratio after 8 weeks of GnRH treatment, and normalization of the lipid profile after estrogen add-back (Al-Omari et al., 1999Go, 2001Go). Gerhard et al. (1992)Go found a significant change in TC and HDL-C levels after 6 months of the same treatment, whereas we previously found significantly enhanced TC, HDL-C and TG levels, but no change in LDL-C in premenopausal women with uterine leiomyomas after six cycles of LAD (Palomba et al., 1998Go). In addition, tibolone add-back, administered for 2 years, prevented analogue-related changes in the lipid profile (Palomba et al., 1999Go). In addition, Cheung et al. (2000)Go reported a significant increase in LDL-C, apo A-1 and apo B after 3 months of LAD treatment. Lastly, TC, TG, HDL-C and LDL-C levels were significantly increased after 3 and 6 months of LAD administration, and were not reverted by ipriflavone administration (Somekawa et al., 2001Go). In contrast, Buhler et al. (1992)Go did not find any variation in lipid metabolism after GnRH analogue treatment.

To evaluate the effect of GnRH analogues administration on lipid, glucose and Hcy metabolism, we excluded from our initial study population (Palomba et al., 2002cGo, dGo) women with a BMI <18 or >30 kg/m2, and with a WHR >0.8 and/or clinical/biochemical hyperandrogenism. In addition, we provide the first anthropometric, hormonal and dietetic assessment in this setting. This is relevant because GnRH analogues have a beneficial metabolic effect in obese and hyperandrogenic women (Efstathiadou and Tsatsoulis, 2001Go).

Our data confirm that GnRH analogue administration alters serum TC, HDL-C, LDL-C and TG levels (Palomba et al., 1998Go; Somekawa et al., 2001Go), and show that the addition of raloxifene does not prevent this effect. In fact, TC, HDL-C and TG levels were significantly increased after treatment with GnRH analogue plus raloxifene. However, the increase in TC and LDL-C concentrations was significantly less in women who received add-on raloxifene than in women who received placebo addition. Indeed, the HDL-C/LDL-C ratio was not changed after GnRH analogue plus raloxifene, which confirms the beneficial effect of raloxifene on the lipid pattern in post-menopausal women (De Leo et al., 2001Go; Walsh, 2001Go).

Fasting insulin and glucose levels are increased in post-menopausal versus premenopausal women, which would imply worsened insulin resistance with menopause (Carr, 2003Go). However, advanced chronological age and central adiposity are two important confounding factors associated with increased insulin resistance (Carr, 2003Go). In this view, even though we did not measure insulin resistance with the euglycemic–hyperinsulinemic clamp procedure, our study should be considered confirmation that severe GnRH analogue-related hypoestrogenism significantly reduces insulin sensitivity. In fact, serum insulin levels and HOMA scores were significantly higher after six cycles of GnRH analogue treatment, and unchanged in the women treated with GnRH analogue plus raloxifene.

In unselected post-menopausal women, raloxifene administration did not affect insulin sensitivity or glucose metabolism (Oleksik et al., 2001Go; Andersson et al., 2002Go; Cagnacci et al., 2002Go; Cucinelli et al., 2002Go). In addition, raloxifene significantly reduced insulin circulating plasma values in hyperinsulinemic women by increasing fractional hepatic insulin extraction and improving peripheral insulin sensitivity (Cucinelli et al., 2002Go).

Our results confirm that circulating Hcy levels are inversely related to blood levels of vitamin B12 and folate (Somekawa et al., 2002Go). The most common cause of abnormal serum Hcy levels in the general population seems to be reduced efficiency of methylenetetrahydrofolate reductase (MTHFR), an enzyme involved in the folate-dependent remethylation of Hcy to methionine (Refsum et al., 1998Go). The high Hcy concentrations observed in women with the thermolabile MTHFR genotype (homozygotes for the causative C677T mutation) are due to a low dietary intake of vitamins and to enhanced susceptibility to this reduced alimentary support (Jacques et al., 2002Go). On the contrary, when folate consumption is high, the thermolabile genotype has little effect on Hcy levels (Refsum et al., 1998Go). Consequently, in order to obtain a homogeneous study group and to eliminate confounding dietary/genetic factors, we enrolled only women with a low folate intake (Food Nutrition Board, National Research Council, 1977Go). Because of the limitations and complexity of procedures for the evaluation of food and nutrient intake (Jacques and Tucker, 2001Go), we used serum folate as a measure of this parameter. Given the lack of correlation between genotype and fasting Hcy in subjects with plasma folate levels at or above the median (15 nmol/l) (Jacques et al., 1996Go), the cut-off we used in the present study was very close, and it was lower than that used by Somekawa et al. (<13.6 nmol/l) (Food Nutrition Board, National Research Council, 1977Go; Somekawa et al., 2002Go).

In our subjects, Hcy levels were significantly increased after 6 months of LAD treatment. Increased Hcy concentrations have already been found in post-menopausal women and probably play a role in the increased incidence of cardiovascular disease observed in post-menopausal women (Madsen et al., 2002Go). Increased serum Hcy level is probably consequent to the altered hormonal status in post-menopause (Hak et al., 2000Go). In fact, hormone replacement reduces serum Hcy concentrations (Mijatovic et al., 1998Go; Somekawa et al., 2002Go). Our finding that raloxifene prevented the increase in serum Hcy levels is in agreement with randomized, controlled clinical trials showing a reduction (varying between 3% and 19%) in serum Hcy levels in post-menopausal women treated with raloxifene (Mijatovic et al., 1998Go; Walsh et al., 2000Go; De Leo et al., 2001Go; Smolders et al., 2002Go).

We found a significant correlation between HOMA score and Hcy levels, which confirms that plasma insulin levels influence Hcy metabolism (Orio et al., 2003Go) by acting on MTHFR and cystathione-{beta}-synthase (Fonseca et al., 2000Go; 2002Go). In premenopausal women, Hcy levels are independently associated with insulin resistance (De Pergola et al., 2001Go), and this association seems to be stronger in hyperinsulinemic obese subjects (Sanchez-Margalet et al., 2002Go). In a large observational study, Meigs et al. (2001)Go confirmed that fasting plasma Hcy levels are directly associated with insulin resistance.

In conclusion, our results demonstrate that in healthy premenopausal women GnRH analogue administration induces a significant increase in serum lipoprotein and Hcy levels, and a significant reduction in insulin sensitivity. These metabolic alterations may be prevented and/or reduced by the addition of raloxifene as add-back therapy.


    Acknowledgements
 
We are grateful to Dr Benito Chinea (Ibis Informatica, Milan, Italy) for statistical support and to Mrs Jean Ann Gilder for help with revising the manuscript.


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 Results
 Discussion
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Submitted on May 6, 2003; resubmitted on July 17, 2003; accepted on October 2, 2003.