- Email: [email protected]
Intranasal 17β-estradiol treatment and Vitamin B12, folate and homocysteine in menopause
Maturitas 50 (2005) 353–358
Intranasal 17-estradiol treatment and Vitamin B12, folate and homocysteine in menopause Muge Harmaa,∗ , Mehmet Harmaa , Abdurrahim Kocyigitb , Tuba Yaltalıa a
Department of Obstetrics and Gynecology, University of Harran, Medical School, Sanliurfa, Turkey b Department of Biochemistry, University of Harran, Medical School, Sanliurfa, Turkey Received 28 April 2004; received in revised form 6 September 2004; accepted 7 September 2004
Abstract Objective: This study assessed the effect of intranasal administration of 17-estradiol (Aerodiol® ) on plasma levels of homocysteine, Vitamin B12 and folate in postmenopausal women. Methods: In all, 26 symptomatic postmenopausal women who had undergone hysterectomy and oophorectomy at least 12 months previously participated in this 6-month randomized prospective clinical study. Menopause was determined by serum FSH level >30 IU/ml and serum estradiol concentration <30 pg/ml. Intranasal 17-estradiol treatment was given once daily at a standard daily dose of 300 g to 16 women, and 10 did not receive any treatment. Results: In the group receiving intranasal 17-estradiol, mean (±S.D.) plasma homocysteine level decreased significantly from pre-treatment values (from 16.68 ± 4.33 to 14.15 ± 1.18 nmol/ml, p = 0.029) and the mean folate level increased (from 4.11 ± 0.80 to 5.64 ± 1.87 ng/ml, p = 0.012). Vitamin B12 levels showed a tendency towards increasing. In the treated group, significant negative correlations were observed between homocysteine and folate values (r = −0.586, p = 0.017) and between homocysteine and Vitamin B12 values (r = −0.672, p = 0.004). No significant changes were observed in the untreated group. Conclusion: The reduction in plasma homocysteine levels observed after 6 months’ treatment with intranasal 17-estradiol may reflect an alteration in folate and Vitamin B12 homeostasis. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Folate; Homocysteine; Intranasal 17-estradiol; Postmenopause; Vitamin B12
1. Introduction
∗ Corresponding author. Present address: 6. Sokak, 2/9, Bahcelievler, 06500, Ankara, Turkey Tel.: +90 414 3163032; fax: +90 414 3163032. E-mail address: [email protected] (M. Harma).
The role of estrogen in altering cardiovascular disease risk in women is contentious [1]. Menopause is associated with increased risk of ischemic heart disease and cerebrovascular disease, which collectively are the main causes of morbidity and mortality in developed nations [1].
0378-5122/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.maturitas.2004.09.002
354
M. Harma et al. / Maturitas 50 (2005) 353–358
The effects of postmenopausal hormone replacement therapy (HRT) on cardiovascular risk markers vary according to the formulation and route of administration, with oral estrogen therapy being the most extensively studied [1]. Homocysteine is the demethylated derivative of the essential amino acid methionine. Plasma concentrations of homocysteine are genetically determined in part, but acquired states such as deficiencies in folic acid and Vitamin B12, and renal failure, can increase blood homocysteine levels [2,3]. Sex hormones influence blood homocysteine levels, but their effects on homocysteine metabolism are not fully understood [4], although, it is generally accepted that homocysteine levels are increased by androgens and decreased by estrogen [5]. In postmenopausal women, hormonal therapy produced decreased fasting blood homocysteine levels in all [6–11] but one study [12]. Intranasal 17-estradiol (Aerodiol® ) is an aqueous solution of natural estradiol delivered by a metered pump, and represents a new approach to the treatment of menopausal symptoms and a new concept of pulsed estrogen therapy. There are no reports investigating the effects of intranasal 17-estradiol on homocysteine metabolism. In the present study, the effects of intranasal 17estradiol on plasma levels of Vitamin B12, folate and homocysteine levels were investigated.
and serum estradiol <30 pg/ml. In all, 26 women were divided into two groups: 16 who used intranasal 17estradiol 300 g once daily and 10 who did not use any treatment for 6 months. The mean age for the treatment group was 52.3 ± 2.67 and for the control group 52.7 ± 2.62 years. 2.2. Samples All participants’ blood samples were withdrawn from the antecubital vein into heparinized tubes between 8 a.m. and 10 a.m. (after fasting from 10 p.m.) and immediately stored on ice at 4 ◦ C. Plasma was separated from the cells by centrifugation at 3000 rpm for 10 min, and the samples were stored at −80 ◦ C until analysis. 2.2.1. Assay for Vitamin B12, folate and homocysteine Plasma Vitamin B12 and folate levels were determined by electrochemiluminescence using commercial kits (Roche, Mannheim, Germany) and with an automatic chemiluminassay analyzer (Elecsys 2010; Elecsys, Germany). Plasma homocysteine levels were determined by chemiluminescence using a commercial kit (DPCImmulite, Los Angeles, USA) and with an automatic chemiluminassay analyzer (Immulite, USA). 2.3. Statistical analysis
2. Materials and methods 2.1. Subjects Approval was obtained from the institutional review board for human subject research, and the ethical standards of the 1983 Declaration of Helsinki were followed. Written informed consent was obtained from all participants. Exclusion criteria were obesity, systemic diseases (such as diabetes and hypertension), smoking, alcohol intake, and use of HRT. Each subject was requested not to take vitamin supplements for the entire period of investigation. This prospective, observational clinical study included symptomatic postmenopausal women volunteers who had previously undergone hysterectomy and oophorectomy. Menopause was determined at least 12 months after operation by serum FSH level >30 IU/ml
The differences between the values before and after treatment were tested within the groups using paired Student’s t-tests. Differences between the changes for the treated and untreated group values were tested using independent Student’s t-tests. Pearson correlation analysis was used to calculate the statistical relationships between Vitamin B12, folate and homocysteine levels between the groups. A P-value < 0.05 was considered statistically significant. All the results are expressed as mean ± S.D. All statistical analyses were performed using SPSS for Windows, version 11.0.
3. Results The clinical characteristics of subjects at baseline (before) and after 6 months’ treatment with intranasal
M. Harma et al. / Maturitas 50 (2005) 353–358
17-estradiol or untreated group are reported in Table 1. At baseline, no significant differences were observed between the two groups. The body mass indices (kg/m2 ) for both groups remained unchanged throughout the trial for both the control and the treated group [(controls: 23.1 ± 1.02 versus 23.2 ± 0.9; difference 0.11 ± 0.38) (treated: 23.2 ± 1.01 versus 22.7 ± 2.06; difference −0.39 ± 0.77) (p = 0.07)]. Changes in the differences between the pre- and post-trial values for the plasma levels of homocysteine, folate, and Vitamin B12 as well as the lipid profiles are summarised in Table 1. Changes in the Vitamin B12 and LDL levels were not significantly different between the two groups. Treatment with intranasal 17-estradiol for 6 months significantly decreased mean fasting homocysteine from 16.68 ± 4.33 to 14.15 ± 1.18 nmol/ml (p = 0.029) and increased mean folate levels from 4.11 ± 0.80 to 5.64 ± 1.87 ng/ml (p = 0.012). Mean
355
plasma Vitamin B12 levels showed a tendency towards increasing with intranasal 17-estradiol treatment but the effect was not statistically significant. Total plasma cholesterol, LDL, HDL and triglycerides were significantly decreased after 6 months’ treatment with intranasal 17-estradiol (Table 1). The decreases in plasma homocysteine levels associated with intranasal 17-estradiol treatment were inversely related to plasma folate and Vitamin B12 levels in the treated group (p = 0.017 and 0.004, respectively) (Figs. 1 and 2).
4. Discussion Intranasal 17-estradiol is a new therapeutic approach for estrogen replacement therapy—an aqueous solution administered by the nasal route, introducing a
Table 1 Characteristics of women evaluated before and after 6 months treatment with intranasal 17-estradiol Parameters
Treated group (n = 16)
Untreated group (n = 10)
pa
16.68 ± 4.33 14.15 ± 1.18 −2.51 ± 2.28*
17.05 ± 5.11 16.10 ± 3.66 −0.61 ± 2.07
0.041
Before After Difference
4.11 ± 0.80 5.64 ± 1.87 1.54 ± 2.25*
5.42 ± 2.47 4.57 ± 1.20 −0.86 ± 0.73
0.004
Vitamin B12 (pg/ml)
Before After Difference
212.11 ± 74.24 256.19 ± 98.74 43.44 ± 62.37
225.24 ± 61.90 227.82 ± 78.95 2.89 ± 74.84
0.148
Triglycerides (mg/dl)
Before After Difference
150.50 ± 67.18 121.68 ± 44.30 −28.81 ± 58.60*
122.20 ± 40.24 140.30 ± 37.14 18.10 ± 34.98
0.032
Total cholesterol (mg/dl)
Before After Difference
193.37 ± 35.06 168.87 ± 28.47 −24.50 ± 18.11**
176.30 ± 40.36 204.40 ± 43.22 28.10 ± 12.68
0.001
HDL (mg/dl)
Before After Difference
47.25 ± 4.55 54.49 ± 2.92 7.24 ± 5.96***
50.24 ± 2.91 50.50 ± 2.67 0.25 ± 4.38
0.004
LDL (mg/dl)
Before After Difference
118.01 ± 28.01 101.50 ± 21.12 −16.5 ± 20.86**
123.00 ± 23.59 117.32 ± 9.65 −5.68 ± 21.02
0.212
Homocysteine (nmol/ml)
Before After Differenceb
Folate (ng/ml)
Results are expressed as mean ± S.D. ∗ p < 0.05. ∗∗ p < 0.01. ∗∗∗ p < 0.001. a Shows the changes between the differences among the groups, analysed using independent Student’s t-tests. b Shows the difference before and after treatment, analysed using paired Student’s t-tests.
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M. Harma et al. / Maturitas 50 (2005) 353–358
Fig. 1. Plasma homocysteine vs. folate levels associated with 6 months’ treatment with intranasal 17-estradiol in postmenopausal women.
new concept of pulsed estrogen therapy. The efficacy of intranasal 17-estradiol in the treatment of menopausal symptoms has been demonstrated in a randomized, double-blind, placebo-controlled trial [13]. At a single dose of 300 g/day, intranasal 17-estradiol has been shown to have equivalent efficacy, as measured
by Kupperman index, to a reference oral estradiol therapy of 2 mg/day, and has shown better gynecological acceptability [14]. Plasma levels of homocysteine, an amino acid derived from methionine metabolism, are reported to be modified by sex hormones [15], and estrogen status
Fig. 2. Plasma homocysteine vs. Vitamin B12 levels associated with 6 months’ treatment with intranasal 17-estradiol in postmenopausal women.
M. Harma et al. / Maturitas 50 (2005) 353–358
is known to affect homocysteine metabolism [5,16]. Because hyperhomocysteinemia is a known factor involved in cardiovascular disease [17,18], it is possible that at least part of the cardioprotective effect of HRT may involve a reduction in blood homocysteine levels [7,10,19]. Earlier studies on the relationship between HRT and homocysteine reported a decrease in blood homocysteine levels in women receiving HRT [6,7,9,20]. However, two long-term studies have shown differing results. Mijatovic et al. [6] found that homocysteine levels were significantly reduced after 24 months’ treatment with conjugated equine estrogen. The postmenopausal Estrogen/Progestin intervention trials, however, reported a modest, significant reduction in plasma homocysteine levels after 12 months of HRT treatment that did not persist over 36 months of treatment [9]. Deficiencies in folic acid and Vitamin B12 are known to increase blood homocysteine levels [2,3]. In earlier studies, oral contraceptives containing ethinyl E2 were shown to decrease Vitamin B12 and folate levels [23]. Serum Vitamin B12 concentrations are frequently low in the elderly, but the cause is not apparent [22]. Carmel et al. [22] proposed that female sex hormones may improve vitamin B metabolism, but this hypothesis was not confirmed convincingly in women using HRT. In the present study we found that plasma homocysteine levels decreased and folate levels increased in the group receiving intranasal 17-estradiol for 6 months. Plasma Vitamin B12 levels showed a tendency towards increasing. Madsen et al. [21] reported a decrease in plasma homocysteine levels with HRT, but found no difference in plasma folate levels. Plasma Vitamin B12 concentrations were slightly, but significantly, lower in the group receiving HRT [21]. Results from the present study provide evidence that HRT-associated alterations in blood homocysteine concentrations may be associated with alterations in folate and Vitamin B12 metabolism. The reduction in plasma homocysteine levels observed after 6 months’ treatment with intranasal 17-estradiol in the present study may reflect alterations in folate and Vitamin B12 homeostasis. In conclusion, short-term treatment with pulsed intranasal 17-estradiol significantly decreased blood homocysteine levels, increased folate levels, and
357
showed a tendency towards increasing Vitamin B12 concentrations in symptomatic surgically menopausal women. It remains to be determined whether these effects influence long term cardiovascular outcomes and vitamin homeostasis in postmenopausal women. The possibility of any therapeutic benefits of nasally administered estradiol over the non-oral route also merits further comparative study. References [1] Davison S, Davis SR. New markers for cardiovascular disease risk in women: impact of endogenous estrogen status and exogenous postmenopausal hormone therapy. J Clin Endocrinol Metab 2003;88:2470–8. [2] Refsum H, Ueland PM, Nygard O, Vollset SE. Homocysteine and cardiovascular disease. Annu Rev Med 1998;49:31–62. [3] Berg K, Malinow MR, Kierulf P, Upson B. Population variation and genetics of plasma homocyst(e)ine level. Clin Genet 1992;41:315–21. [4] Cagnacci A, Malmusi S, Zanni AL, Alessandrini C, Caretto S, Volpe A. Comparison of the effect of oral and transdermal hormone therapy on fasting and postmethionine homocysteine levels. Fertil Steril 2004;81:99–103. [5] Giltay EJ, Hoogeveen EK, Elbers JM, Gooren LJ, Asscheman H, Stehouwer CD. Effects of sex steroids on plasma total homocysteine levels: a study in transsexual males and females. J Clin Endocrinol Metab 1998;83:550–3. [6] Mijatovic V, Kenemans P, Netelenbos C, et al. Postmenopausal oral 17-estradiol continuously combined with dydrogesterone reduces fasting serum homocysteine levels. Fertil Steril 1998;69:876–82. [7] van der Mooren MJ, Wouters MG, Blom HJ, Schellekens LA, Eskes TK, Rolland R. Hormone replacement therapy may reduce high serum homocysteine in postmenopausal women. Eur J Clin Invest 1994;24:733–6. [8] van Baal WM, Smolders RG, van der Mooren MJ, Teerlink T, Kenemans P. Hormone replacement therapy and plasma homocysteine levels. Obstet Gynecol 1999;94:485–91. [9] Barnabei VM, Phillips TM, Hsia J. Plasma homocysteine in women taking hormone replacement therapy: the postmenopausal Estrogen/Progestin ınterventions (PEPI) trial. J Womens Health Gend Based Med 1999;8:1167–72. [10] Walsh BW, Paul S, Wild RA, et al. The effects of hormone replacement therapy and raloxifene on C-reactive protein and homocysteine in healthy postmenopausal women: a randomized, controlled trial. J Clin Endocrinol Metab 2000;85:214–8. [11] Ventura P, Cagnacci A, Malmusi S, et al. Continuous combined hormone replacement therapy with oral 17-estradiol and norethisterone acetate improves homocysteine metabolism in postmenopausal women. Menopause 2001;8:252–8. [12] Evio S, Tiitinen A, Turpeinen U, Ylikorkala O. Failure of the combination of sequential oral and transdermal estradiol plus norethisterone acetate to affect plasma homocysteine levels. Fertil Steril 2000;74:1080–3.
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[13] Studd J, Pornel B, Marton I, et al. Efficacy and acceptability of intranasal 17 beta-oestradiol for menopausal symptoms: randomised dose-response study. Aerodiol Study Group Lancet 1999;353:1574–8. [14] Mattsson LA, Christiansen C, Colau JC, et al. Clinical equivalence of intranasal and oral 17-estradiol for postmenopausal symptoms. Am J Obstet Gynecol 2000;182:545– 52. [15] Marchesoni D, Driul L, Plaino L, Villani MT, Becagli L, Mozzanega B. Menopause rather than estrogen modifies plasma homocysteine levels. Int J Gynaecol Obstet 2003;81: 293–7. [16] Wouters MG, Moorrees MT, van der Mooren MJ, et al. Plasma homocysteine and menopausal status. Eur J Clin Invest 1995;25:801–5. [17] Graham IM, Daly LE, Refsum HM, et al. Plasma homocysteine as a risk factor for vascular disease. The European Concerted Action Project. JAMA 1997;277:1775–81. [18] Kuller LH, Evans RW. Homocysteine, vitamins, and cardiovascular disease. Circulation 1998;98:196–9.
[19] Mijatovic V, Kenemans P, Jakobs C, van Baal WM, PetersMuller ER, van der Mooren MJ. A randomized controlled study of the effects of 17-estradiol-dydrogesterone on plasma homocysteine in postmenopausal women. Obstet Gynecol 1998;91:432–6. [20] van der Mooren MJ, Demacker PN, Blom HJ, de Rijke YB, Rolland R. The effect of sequential three-monthly hormone replacement therapy on several cardiovascular risk estimators in postmenopausal women. Fertil Steril 1997;67:67–73. [21] Madsen JS, Kristensen SR, Klitgaard NA, et al. Effect of longterm hormone replacement therapy on plasma homocysteine in postmenopausal women: a randomized controlled study. Am J Obstet Gynecol 2002;187:33–9. [22] Carmel R, Howard JM, Green R, Jacobsen DW, Azen C. Hormone replacement therapy and cobalamin status in elderly women. Am J Clin Nutr 1996;64:856–9. [23] Steegers-Theunissen RP, Boers GH, Steegers EA, Trijbels FJ, Thomas CM, Eskes TK. Effects of sub-50 oral contraceptives on homocysteine metabolism: a preliminary study. Contraception 1992;45:129–39.
Intranasal 17-estradiol treatment and Vitamin B12, folate and homocysteine in menopause Muge Harmaa,∗ , Mehmet Harmaa , Abdurrahim Kocyigitb , Tuba Yaltalıa a
Department of Obstetrics and Gynecology, University of Harran, Medical School, Sanliurfa, Turkey b Department of Biochemistry, University of Harran, Medical School, Sanliurfa, Turkey Received 28 April 2004; received in revised form 6 September 2004; accepted 7 September 2004
Abstract Objective: This study assessed the effect of intranasal administration of 17-estradiol (Aerodiol® ) on plasma levels of homocysteine, Vitamin B12 and folate in postmenopausal women. Methods: In all, 26 symptomatic postmenopausal women who had undergone hysterectomy and oophorectomy at least 12 months previously participated in this 6-month randomized prospective clinical study. Menopause was determined by serum FSH level >30 IU/ml and serum estradiol concentration <30 pg/ml. Intranasal 17-estradiol treatment was given once daily at a standard daily dose of 300 g to 16 women, and 10 did not receive any treatment. Results: In the group receiving intranasal 17-estradiol, mean (±S.D.) plasma homocysteine level decreased significantly from pre-treatment values (from 16.68 ± 4.33 to 14.15 ± 1.18 nmol/ml, p = 0.029) and the mean folate level increased (from 4.11 ± 0.80 to 5.64 ± 1.87 ng/ml, p = 0.012). Vitamin B12 levels showed a tendency towards increasing. In the treated group, significant negative correlations were observed between homocysteine and folate values (r = −0.586, p = 0.017) and between homocysteine and Vitamin B12 values (r = −0.672, p = 0.004). No significant changes were observed in the untreated group. Conclusion: The reduction in plasma homocysteine levels observed after 6 months’ treatment with intranasal 17-estradiol may reflect an alteration in folate and Vitamin B12 homeostasis. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Folate; Homocysteine; Intranasal 17-estradiol; Postmenopause; Vitamin B12
1. Introduction
∗ Corresponding author. Present address: 6. Sokak, 2/9, Bahcelievler, 06500, Ankara, Turkey Tel.: +90 414 3163032; fax: +90 414 3163032. E-mail address: [email protected] (M. Harma).
The role of estrogen in altering cardiovascular disease risk in women is contentious [1]. Menopause is associated with increased risk of ischemic heart disease and cerebrovascular disease, which collectively are the main causes of morbidity and mortality in developed nations [1].
0378-5122/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.maturitas.2004.09.002
354
M. Harma et al. / Maturitas 50 (2005) 353–358
The effects of postmenopausal hormone replacement therapy (HRT) on cardiovascular risk markers vary according to the formulation and route of administration, with oral estrogen therapy being the most extensively studied [1]. Homocysteine is the demethylated derivative of the essential amino acid methionine. Plasma concentrations of homocysteine are genetically determined in part, but acquired states such as deficiencies in folic acid and Vitamin B12, and renal failure, can increase blood homocysteine levels [2,3]. Sex hormones influence blood homocysteine levels, but their effects on homocysteine metabolism are not fully understood [4], although, it is generally accepted that homocysteine levels are increased by androgens and decreased by estrogen [5]. In postmenopausal women, hormonal therapy produced decreased fasting blood homocysteine levels in all [6–11] but one study [12]. Intranasal 17-estradiol (Aerodiol® ) is an aqueous solution of natural estradiol delivered by a metered pump, and represents a new approach to the treatment of menopausal symptoms and a new concept of pulsed estrogen therapy. There are no reports investigating the effects of intranasal 17-estradiol on homocysteine metabolism. In the present study, the effects of intranasal 17estradiol on plasma levels of Vitamin B12, folate and homocysteine levels were investigated.
and serum estradiol <30 pg/ml. In all, 26 women were divided into two groups: 16 who used intranasal 17estradiol 300 g once daily and 10 who did not use any treatment for 6 months. The mean age for the treatment group was 52.3 ± 2.67 and for the control group 52.7 ± 2.62 years. 2.2. Samples All participants’ blood samples were withdrawn from the antecubital vein into heparinized tubes between 8 a.m. and 10 a.m. (after fasting from 10 p.m.) and immediately stored on ice at 4 ◦ C. Plasma was separated from the cells by centrifugation at 3000 rpm for 10 min, and the samples were stored at −80 ◦ C until analysis. 2.2.1. Assay for Vitamin B12, folate and homocysteine Plasma Vitamin B12 and folate levels were determined by electrochemiluminescence using commercial kits (Roche, Mannheim, Germany) and with an automatic chemiluminassay analyzer (Elecsys 2010; Elecsys, Germany). Plasma homocysteine levels were determined by chemiluminescence using a commercial kit (DPCImmulite, Los Angeles, USA) and with an automatic chemiluminassay analyzer (Immulite, USA). 2.3. Statistical analysis
2. Materials and methods 2.1. Subjects Approval was obtained from the institutional review board for human subject research, and the ethical standards of the 1983 Declaration of Helsinki were followed. Written informed consent was obtained from all participants. Exclusion criteria were obesity, systemic diseases (such as diabetes and hypertension), smoking, alcohol intake, and use of HRT. Each subject was requested not to take vitamin supplements for the entire period of investigation. This prospective, observational clinical study included symptomatic postmenopausal women volunteers who had previously undergone hysterectomy and oophorectomy. Menopause was determined at least 12 months after operation by serum FSH level >30 IU/ml
The differences between the values before and after treatment were tested within the groups using paired Student’s t-tests. Differences between the changes for the treated and untreated group values were tested using independent Student’s t-tests. Pearson correlation analysis was used to calculate the statistical relationships between Vitamin B12, folate and homocysteine levels between the groups. A P-value < 0.05 was considered statistically significant. All the results are expressed as mean ± S.D. All statistical analyses were performed using SPSS for Windows, version 11.0.
3. Results The clinical characteristics of subjects at baseline (before) and after 6 months’ treatment with intranasal
M. Harma et al. / Maturitas 50 (2005) 353–358
17-estradiol or untreated group are reported in Table 1. At baseline, no significant differences were observed between the two groups. The body mass indices (kg/m2 ) for both groups remained unchanged throughout the trial for both the control and the treated group [(controls: 23.1 ± 1.02 versus 23.2 ± 0.9; difference 0.11 ± 0.38) (treated: 23.2 ± 1.01 versus 22.7 ± 2.06; difference −0.39 ± 0.77) (p = 0.07)]. Changes in the differences between the pre- and post-trial values for the plasma levels of homocysteine, folate, and Vitamin B12 as well as the lipid profiles are summarised in Table 1. Changes in the Vitamin B12 and LDL levels were not significantly different between the two groups. Treatment with intranasal 17-estradiol for 6 months significantly decreased mean fasting homocysteine from 16.68 ± 4.33 to 14.15 ± 1.18 nmol/ml (p = 0.029) and increased mean folate levels from 4.11 ± 0.80 to 5.64 ± 1.87 ng/ml (p = 0.012). Mean
355
plasma Vitamin B12 levels showed a tendency towards increasing with intranasal 17-estradiol treatment but the effect was not statistically significant. Total plasma cholesterol, LDL, HDL and triglycerides were significantly decreased after 6 months’ treatment with intranasal 17-estradiol (Table 1). The decreases in plasma homocysteine levels associated with intranasal 17-estradiol treatment were inversely related to plasma folate and Vitamin B12 levels in the treated group (p = 0.017 and 0.004, respectively) (Figs. 1 and 2).
4. Discussion Intranasal 17-estradiol is a new therapeutic approach for estrogen replacement therapy—an aqueous solution administered by the nasal route, introducing a
Table 1 Characteristics of women evaluated before and after 6 months treatment with intranasal 17-estradiol Parameters
Treated group (n = 16)
Untreated group (n = 10)
pa
16.68 ± 4.33 14.15 ± 1.18 −2.51 ± 2.28*
17.05 ± 5.11 16.10 ± 3.66 −0.61 ± 2.07
0.041
Before After Difference
4.11 ± 0.80 5.64 ± 1.87 1.54 ± 2.25*
5.42 ± 2.47 4.57 ± 1.20 −0.86 ± 0.73
0.004
Vitamin B12 (pg/ml)
Before After Difference
212.11 ± 74.24 256.19 ± 98.74 43.44 ± 62.37
225.24 ± 61.90 227.82 ± 78.95 2.89 ± 74.84
0.148
Triglycerides (mg/dl)
Before After Difference
150.50 ± 67.18 121.68 ± 44.30 −28.81 ± 58.60*
122.20 ± 40.24 140.30 ± 37.14 18.10 ± 34.98
0.032
Total cholesterol (mg/dl)
Before After Difference
193.37 ± 35.06 168.87 ± 28.47 −24.50 ± 18.11**
176.30 ± 40.36 204.40 ± 43.22 28.10 ± 12.68
0.001
HDL (mg/dl)
Before After Difference
47.25 ± 4.55 54.49 ± 2.92 7.24 ± 5.96***
50.24 ± 2.91 50.50 ± 2.67 0.25 ± 4.38
0.004
LDL (mg/dl)
Before After Difference
118.01 ± 28.01 101.50 ± 21.12 −16.5 ± 20.86**
123.00 ± 23.59 117.32 ± 9.65 −5.68 ± 21.02
0.212
Homocysteine (nmol/ml)
Before After Differenceb
Folate (ng/ml)
Results are expressed as mean ± S.D. ∗ p < 0.05. ∗∗ p < 0.01. ∗∗∗ p < 0.001. a Shows the changes between the differences among the groups, analysed using independent Student’s t-tests. b Shows the difference before and after treatment, analysed using paired Student’s t-tests.
356
M. Harma et al. / Maturitas 50 (2005) 353–358
Fig. 1. Plasma homocysteine vs. folate levels associated with 6 months’ treatment with intranasal 17-estradiol in postmenopausal women.
new concept of pulsed estrogen therapy. The efficacy of intranasal 17-estradiol in the treatment of menopausal symptoms has been demonstrated in a randomized, double-blind, placebo-controlled trial [13]. At a single dose of 300 g/day, intranasal 17-estradiol has been shown to have equivalent efficacy, as measured
by Kupperman index, to a reference oral estradiol therapy of 2 mg/day, and has shown better gynecological acceptability [14]. Plasma levels of homocysteine, an amino acid derived from methionine metabolism, are reported to be modified by sex hormones [15], and estrogen status
Fig. 2. Plasma homocysteine vs. Vitamin B12 levels associated with 6 months’ treatment with intranasal 17-estradiol in postmenopausal women.
M. Harma et al. / Maturitas 50 (2005) 353–358
is known to affect homocysteine metabolism [5,16]. Because hyperhomocysteinemia is a known factor involved in cardiovascular disease [17,18], it is possible that at least part of the cardioprotective effect of HRT may involve a reduction in blood homocysteine levels [7,10,19]. Earlier studies on the relationship between HRT and homocysteine reported a decrease in blood homocysteine levels in women receiving HRT [6,7,9,20]. However, two long-term studies have shown differing results. Mijatovic et al. [6] found that homocysteine levels were significantly reduced after 24 months’ treatment with conjugated equine estrogen. The postmenopausal Estrogen/Progestin intervention trials, however, reported a modest, significant reduction in plasma homocysteine levels after 12 months of HRT treatment that did not persist over 36 months of treatment [9]. Deficiencies in folic acid and Vitamin B12 are known to increase blood homocysteine levels [2,3]. In earlier studies, oral contraceptives containing ethinyl E2 were shown to decrease Vitamin B12 and folate levels [23]. Serum Vitamin B12 concentrations are frequently low in the elderly, but the cause is not apparent [22]. Carmel et al. [22] proposed that female sex hormones may improve vitamin B metabolism, but this hypothesis was not confirmed convincingly in women using HRT. In the present study we found that plasma homocysteine levels decreased and folate levels increased in the group receiving intranasal 17-estradiol for 6 months. Plasma Vitamin B12 levels showed a tendency towards increasing. Madsen et al. [21] reported a decrease in plasma homocysteine levels with HRT, but found no difference in plasma folate levels. Plasma Vitamin B12 concentrations were slightly, but significantly, lower in the group receiving HRT [21]. Results from the present study provide evidence that HRT-associated alterations in blood homocysteine concentrations may be associated with alterations in folate and Vitamin B12 metabolism. The reduction in plasma homocysteine levels observed after 6 months’ treatment with intranasal 17-estradiol in the present study may reflect alterations in folate and Vitamin B12 homeostasis. In conclusion, short-term treatment with pulsed intranasal 17-estradiol significantly decreased blood homocysteine levels, increased folate levels, and
357
showed a tendency towards increasing Vitamin B12 concentrations in symptomatic surgically menopausal women. It remains to be determined whether these effects influence long term cardiovascular outcomes and vitamin homeostasis in postmenopausal women. The possibility of any therapeutic benefits of nasally administered estradiol over the non-oral route also merits further comparative study. References [1] Davison S, Davis SR. New markers for cardiovascular disease risk in women: impact of endogenous estrogen status and exogenous postmenopausal hormone therapy. J Clin Endocrinol Metab 2003;88:2470–8. [2] Refsum H, Ueland PM, Nygard O, Vollset SE. Homocysteine and cardiovascular disease. Annu Rev Med 1998;49:31–62. [3] Berg K, Malinow MR, Kierulf P, Upson B. Population variation and genetics of plasma homocyst(e)ine level. Clin Genet 1992;41:315–21. [4] Cagnacci A, Malmusi S, Zanni AL, Alessandrini C, Caretto S, Volpe A. Comparison of the effect of oral and transdermal hormone therapy on fasting and postmethionine homocysteine levels. Fertil Steril 2004;81:99–103. [5] Giltay EJ, Hoogeveen EK, Elbers JM, Gooren LJ, Asscheman H, Stehouwer CD. Effects of sex steroids on plasma total homocysteine levels: a study in transsexual males and females. J Clin Endocrinol Metab 1998;83:550–3. [6] Mijatovic V, Kenemans P, Netelenbos C, et al. Postmenopausal oral 17-estradiol continuously combined with dydrogesterone reduces fasting serum homocysteine levels. Fertil Steril 1998;69:876–82. [7] van der Mooren MJ, Wouters MG, Blom HJ, Schellekens LA, Eskes TK, Rolland R. Hormone replacement therapy may reduce high serum homocysteine in postmenopausal women. Eur J Clin Invest 1994;24:733–6. [8] van Baal WM, Smolders RG, van der Mooren MJ, Teerlink T, Kenemans P. Hormone replacement therapy and plasma homocysteine levels. Obstet Gynecol 1999;94:485–91. [9] Barnabei VM, Phillips TM, Hsia J. Plasma homocysteine in women taking hormone replacement therapy: the postmenopausal Estrogen/Progestin ınterventions (PEPI) trial. J Womens Health Gend Based Med 1999;8:1167–72. [10] Walsh BW, Paul S, Wild RA, et al. The effects of hormone replacement therapy and raloxifene on C-reactive protein and homocysteine in healthy postmenopausal women: a randomized, controlled trial. J Clin Endocrinol Metab 2000;85:214–8. [11] Ventura P, Cagnacci A, Malmusi S, et al. Continuous combined hormone replacement therapy with oral 17-estradiol and norethisterone acetate improves homocysteine metabolism in postmenopausal women. Menopause 2001;8:252–8. [12] Evio S, Tiitinen A, Turpeinen U, Ylikorkala O. Failure of the combination of sequential oral and transdermal estradiol plus norethisterone acetate to affect plasma homocysteine levels. Fertil Steril 2000;74:1080–3.
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