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HDL concentration, lipid transfer to HDL, and HDL size in normolipidemic nonobese menopausal women
International Journal of Gynecology and Obstetrics 104 (2009) 117–120
Contents lists available at ScienceDirect
International Journal of Gynecology and Obstetrics j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i j g o
CLINICAL ARTICLE
HDL concentration, lipid transfer to HDL, and HDL size in normolipidemic nonobese menopausal women Arícia H. Giribela a, Nilson R. Melo a, Maria C. Latrilha b, Edmundo C. Baracat a, Raul C. Maranhão b,c,⁎ a b c
Gynecological Department and Lipid Metabolism Laboratory, University of Sao Paulo, São Paulo, Brazil Heart Institute (INCOR) of the School of Medicine, University of Sao Paulo, São Paulo, Brazil Faculty of Pharmaceutical Sciences, University of Sao Paulo, São Paulo, Brazil
a r t i c l e
i n f o
Article history: Received 20 June 2008 Received in revised form 15 September 2008 Accepted 1 October 2008 Keywords: Aging Cholesterol Estrogen High-density lipoproteins Lipid transfer Menopause Triglycerides
a b s t r a c t Objective: To determine the impact of menopause on lipid transfer from donor lipoproteins to high-density lipoproteins (HDLs)—a process that is related to the protective function of HDL—and the size of HDL particles. Method: Plasma from 22 premenopausal and 18 postmenopausal nonobese, normolipidemic women paired for age (40–50 years) was incubated in an artificial nanoemulsion labeled with radioactive lipids. Then the HDL fraction was assessed for radioactivity; the percentage of radioactive lipids transferred from the nanoemulsion to HDL was determined; and the size of HDL particles was measured by laser light scattering. Results: There were no differences between the 2 groups in serum concentration of HDL cholesterol (61 ± 12 mg/dL vs 61 ± 14 mg/dL) or apolipoprotein A1 (1.5 ± 0.3 g/L vs 1.5 ± 0.2 g/L); lipid transfer to HDL; or size of HDL particles (8.8 ± 0.8 vs 9.0 ± 0.5 nm). Conclusion: Menopause was not found to affect HDL cholesterol plasma concentration, lipid transfer to HDL, or size of HDL particles in normolipidemic nonobese women. © 2008 International Federation of Gynecology and Obstetrics. Published by Elsevier Ireland Ltd. All rights reserved.
1. Introduction For those affected, coronary artery disease (CAD) typically develops 10 to 15 years later in women than in men [1]. As the plasma concentration of low-density lipoprotein cholesterol (LDL-C) is positively correlated with CAD, and the plasma concentration of highdensity lipoprotein cholesterol (HDL-C) is negatively correlated with CAD, plasma lipid status is one of the cardinal factors involved in atherogenesis [2]. In women, LDL-C and HDL-C levels are, respectively, lower and higher than in men, and lower HDL-C levels are more consistently predictive of the appearance of CAD symptoms [3]. Menopause is generally considered a turning point for the risk of CAD [3]. The delay in CAD development in women is attributed to hormonal protection before menopause, which, it is argued, is lost after ovarian hormone production ceases [1]. Fukami et al. [4] showed that the metabolic changes take place within the year preceding menopause. Most studies show that LDL-C concentration rises after menopause and Mathews et al. [3] observed that it rises sharply within 6 months of the cessation of menses. Decreasing estrogen levels are associated with a decrease in LDL-C removal from blood plasma, a condition that hormone treatment can revert [5,6].
In contrast, the effect of menopause on HDL-C is uncertain. There have been reports of increased [7], decreased [8,9], or sustained [8,9] levels; moreover, Mercuro et al. [10] reported that after menopause, concurrently with a gradual decrease in HDL-C concentration over 2 years, there was a rise in apolipoprotein A1 levels, which implies a possible change in HDL composition. Aging is the prominent factor in the appearance of CAD. In both sexes, the aging process results in a diminution of the removal of LDL-C from blood plasma, resulting in increased LDL-C levels. As the effect of age on HDL-C is as controversial as that of menopause, the concomitant—even synergistic—effects of aging and menopause on plasma lipids should be rigorously set apart [11]. By selecting premenopausal and postmenopausal women of the same age range, this cross-sectional study evaluated exclusively the impact of menopause on plasma lipid concentration, lipid transfer to HDL, and size of HDL particles. To avoid the interference of elevated serum levels of LDL-C and triglycerides on HDL metabolism, only women with serum cholesterol levels less than 200 mg/dL and triglyceride levels less than 150 mg/dL were included. The similar levels of those lipids in all participants are therefore due to the inclusion criteria. 2. Materials and methods
⁎ Corresponding author. Instituto do Coração (InCor) do Hospital das Clínicas da USP, Laboratório de Metabolismo de Lípides, Av. Dr. Enéas C. Aguiar 44, 1o Subsolo, CEP05403-000, São Paulo-SP, Brazil. Fax: +55 11 30695574. E-mail address: [email protected] (R.C. Maranhão).
A total of 22 consecutive premenopausal and 18 postmenopausal healthy women were enrolled at the Gynecological Department of Clinics Hospital of the University of São Paulo, where they were
0020-7292/$ – see front matter © 2008 International Federation of Gynecology and Obstetrics. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijgo.2008.10.001
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A.H. Giribela et al. / International Journal of Gynecology and Obstetrics 104 (2009) 117–120
receiving periodic checkups. They were aged between 40 and 50 years and had a total cholesterol level less than 200 mg/dL, a LDL-C level less than 120 mg/dL, and a triglyceride level less than 150 mg/dL. Exclusion criteria were obesity, defined as a body mass index (BMI, calculated as weight in kilograms divided by the square of height in meters) greater than 30; habitual consumption of tobacco or alcohol; apparent or reported cardiovascular disease; diabetes and/or other metabolic disease; hypertension; cancer; pregnancy or breastfeeding; and use of contraceptives, hormone therapy, or antihyperlipidemic drugs. No participant had a history of any disease known to affect lipid metabolism. None was engaging in regular training exercises and all followed a typical local diet, without dietary restrictions. All had normal plasma levels of thyroid stimulating hormone, tri-iodothyronine, and free thyroxine. Menopausal status was defined by the presence or absence of amenorrhea and elevated plasma levels of follicle-stimulating hormone (FSH). The participants were considered postmenopausal if their last period occurred more than 1 year before study entry and their FSH plasma level was greater than 30 mUI/mL. They were considered premenopausal if they had regular cycles, no menopauserelated symptoms, and a FSH level less than 30 mUI/mL. Physical data of the participants are shown in Table 1. The study objective and design were explained to each participant, and all gave written informed consent. The study was approved by the Scientific and Ethics Committee of the University of São Paulo Medical School Hospital. Blood samples were collected by venipuncture after a 12-hour fast. Commercial enzymatic kits were used for the determination of plasma levels of glucose (Boehringer Mannheim, Penzberg, Germany), total cholesterol (Boehringer Mannheim), and triglycerides (Abbott Laboratories, Abbott Park, IL, USA). Levels of HDL-C were determined by the method used for total cholesterol after lipoprotein precipitation with magnesium phosphotungstate; levels of LDL-C were calculated by the Friedewald formula. Apolipoproteins A1 and B were determined by radial immunodiffusion (Boehringer Mannheim). As described previously [12], the size of HDL particles was measured by laser light scattering in the plasma supernatant after chemical precipitation of the lipoproteins containing apolipoprotein B. We used a ZetaPALS Zeta Potential analyzer (Brookhaven Intruments, Holtsville, NY, USA). The nanoemulsion contained a lipid mixture composed of 40 mg of cholesteryl oleate, 20 mg of egg phosphaditylcholine, 1 mg of triolein, and 0.5 mg of cholesterol (all from Sigma-Aldrich, St Louis, MO, USA), to which were added the radioactive lipids (Amersham International, Amersham, UK). It was obtained by the Ginsburg method [13]
Table 1 Physical characteristics, plasma glucose, lipids and apolipoproteins of the study subjects a Variable
Premenopausal group (n = 22)
Postmenopausal group (n = 18)
P value
Age, y Weight, kg Height, m BMI Abdominal circumference, cm Fasting glucose, mg/dL Cholesterol, mg/dL Total HDL-C LDL-C Triglycerides, mg/dL Apolipoprotein A1, g/L Apolipoprotein B, g/L
45 ± 3 61.5 ± 8.2 1.60 ± 0.10 24.9 ± 2.6 81.0 ± 10.4
48 ± 3 62.8 ± 8.3 1.60 ± 0.10 24.5 ± 3.1 84.1 ± 9.0
b 0.01 NS NS NS NS
86 ± 9.1
86 ± 6.8
NS
191 ± 27 61 ± 12 103 ± 35 103 ± 50 1.5 ± 0.33 0.9 ± 0.22
178 ± 24 61± 14 100 ± 24 80 ± 44 1.5 ± 0.22 0.8 ± 0.20
NS NS NS NS NS NS
Abbreviations: BMI, body mass index (calculated as weight in kilograms divided by the square of height in meters); HDL-C, high-density lipoprotein cholesterol; LDL-C, lowdensity lipoprotein cholesterol; NS, not significant. a Values are given as mean ± SD unless otherwise indicated.
Table 2 HDL size and lipid transfer from the nanoemulsion to HDLa Variable Diameter, nm Lipid transfer, % b Free cholesterol Phospholipids Esterified cholesterol Triglycerides
Premenopausal group (n = 22)
Postmenopausal group (n = 18)
P value
9.0 ± 0.5
8.8 ± 0.8
NS
5.6 ± 0.9 17.8 ± 2.1 3.1 ± 1.7 3.6 ± 0.7
5.3 ± 0.8 18.8 ± 1.9 2.7 ± 0.8 3.6 ± 0.9
NS NS NS NS
a
Values are given as mean ± SD. The value indicated is the percentage of the radioactivity of each lipid component in the nanoemulsion that was transferred to the plasma HDL fraction after a 60-minute incubation. The ability of the HDL fraction to receive lipids from LDL, such as free cholesterol, cholesteryl esters, phospholipids, and triglycerides, was measured. This process depends on transfer proteins, which mediate the transfer of lipids from one lipoprotein class to another, and also on the composition and structure of the recipient HDL. Additionally, we evaluated the HDL particle size.
b
modified by Maranhão et al. [14], where lipids are emulsified in an aqueous medium by prolonged ultrasonic irradiation, and the crude emulsion then ultracentrifugated in a 2-step process, with density adjustment by addition of potassium bromide. Two sets of the nanoemulsion were prepared, one labeled with 3H-cholesteryl oleate and 14C-cholesterol and the other with 14C-phosphatidylcholine and 3H-triolein. We then assessed the transfer to plasma HDL of the radioactive lipids present in the nanoemulsion. Plasma was obtained from centrifugating, at 2500 rpm and 4 °C for 15-minutes, blood samples collected into glass tubes containing 0.15% Na2 EDTA. Tubes containing 0.2 mL of plasma and 0.05 mL of a nanoemulsion labeled with either 3 H-cholesteryl oleate and 14C-cholesterol or 14C-phosphatidylcholine and 3H-triolein were placed in a shaking bath and incubated for 60 minutes at 37 °C. Then, 0.25 mL of a solution containing 0.02% of dextran sulfate (molecular weight, 50 000) and 0.3 mol/L of magnesium chloride was added to the tube and vortexed for 30 seconds. Samples were centrifuged at 3000 rpm for 10 minutes and 0.25 mL of the obtained supernatant, which contained the plasma HDL fraction, were transferred to vials containing a scintillation solution, vigorously shaken, and left to stand for at least 45 minutes. Radioactivity was determined using a Packard 1660 TR liquid scintillation counter (Packard BioScience, Meriden, Connecticut, USA). The transfer of the nanoemulsion lipids to HDL was evaluated as percentage of the radioactivity of a given lipid formerly in the nanoemulsion, and found in the plasma HDL fraction after the 60minute incubation period. The statistical differences between the 2 groups were evaluated using the Graf Pad Instat software, version 3.0 (Graf Pad Software, San Diego, CA, USA), by the t test because the groups showed normal distribution. Data were expressed as mean ± standard deviation. P b 0.05 was considered significant in all analyses. 3. Results The participants were slightly older in the postmenopausal than in the premenopausal group (Table 1), but BMI and plasma glucose concentration were similar in the 2 groups. There were no differences between the 2 groups in plasma concentrations of total cholesterol, LDL-C, HDL-C, triglycerides, apolipoprotein A1, or apolipoprotein B. The diameter of HDL particles was similar in the 2 groups (Table 2). Table 2 also shows the results of the assay by which the transfer of free and esterified cholesterol, triglycerides, and phospholipids from the nanoemulsion to HDL was evaluated. There were no differences between the 2 groups regarding the percentage of acceptance by HDL of any of the 4 types of lipids, whether the values were obtained from the entire HDL fraction or from the normalized HDL-C concentration.
A.H. Giribela et al. / International Journal of Gynecology and Obstetrics 104 (2009) 117–120
No correlation was found between the lipid values obtained in this study and BMI, abdominal circumference, or mean group age. In the postmenopausal group the lipid values did not correlate with time elapsed since menopause. 4. Discussion In this study, only women with serum fasting cholesterol less than 200 mg/dL and triglycerides less than 150 mg/dL were included. The similar levels of those lipids in the premenopausal and the postmenopausal group are therefore due to the inclusion criteria and cannot be interpreted as a lack of effect of menopause on plasma concentration of LDL-C or on triglyceride-rich lipoproteins. The see-saw effect of triglyceride levels on HDL is well known, by which HDL levels decrease as triglyceride levels increase, and reciprocally [15]. Since the metabolisms of the different classes of plasma lipoprotein are intertwined, by including only women with total cholesterol and triglyceride less than the cut-off values for risk of CAD, the effects on HDL of alterations of lipoprotein from other classes— such as LDL or very-low-density lipoproteins (VLDL)—were avoided. Therefore, because of the exclusion criteria used to distinguish the primary effects of menopause on HDL metabolism, the interpretation of our results cannot be extended to women who gain weight or become hyperlipidemic during or following menopause. Because of the effects of age on lipid plasma levels and the regulation of lipoprotein metabolism, another criterion in the selection of the participants was age. The participants were about 3 years older in the postmenopausal group, but it did not influence the results. Despite the limitations inherent in cross-sectional studies, the fact that there were no differences between the 2 groups in any of the parameters measured suggests that the hormonal changes occurring during menopause did not influence those parameters. One of the HDL functions in plasma lipid regulation is reverse transport of cholesterol. In reverse transport, HDL removes cholesterol from the peripheral tissues and transports it to the liver, where it is excreted in the bile. Alternatively HDL may transfer cholesterol to lipoproteins from other classes, but the fate of the transferred cholesterol will be the same. The transfer proteins therefore play a major role in the cholesterol removal process. Cholesteryl ester transfer protein (CETP) and phospholipid transfer protein (PLTP) are the major proteins involved in those lipid shifts. The former specifically mediates the transfer of triglycerides from VLDL to LDL and HDL, as well as the transfer of cholesterol esters from LDL and HDL to VLDL. It is associated with HDL in the plasma. As for PLTP, it is partially associated with HDL and mediates the transfer of phospholipids from VLDL to HDL, as well as the reciprocal exchange of phospholipids between VLDL and HDL. Although lipid transfers between lipoproteins of different classes are bidirectional, they frequently result in lipid enrichment or depletion in a given class [16]. It has been increasingly perceived that not only HDL plasma concentration but also the qualitative and functional aspects of HDL are important for its antiatherogenic actions, as is exemplified by HDL containing the Milano mutation of apolipoprotein A1. Even with low levels of circulating HDL, individuals carrying this apolipoprotein A1 variant are protected from cardiovascular events. Similarly, individuals with high levels of HDL that are prone to modification could be less protected than others with lesser levels of HDL that are more resistant to modification [17]. The exchange of lipids to and from HDL particles is crucial to reverse cholesterol transport, and it clearly requires a coordinated metabolic regulation of the HDL subclasses. In recent years, it has been considered that changes in the distribution of HDL subclasses of smaller or larger particles were more closely correlated with atherosclerosis than low plasma levels of HDL-C. Some studies have reported that small-size HDL particles are associated with an increased risk of coronary heart disease, whereas
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large-size HDL particles are associated with a decreased risk [18]. The lack of effect of menopause on particle size in the HDL fraction in the present study suggests that menopause does not change the HDL subclass profile. In this respect, Ushiroyama et al. [19] and Stevenson et al. [8] reported that there were fewer HDL2 (larger) particles and more HDL3 (smaller) particles in the plasma of postmenopausal women, compared with premenopausal women. However, this finding can be attributed to increased triglyceride levels in the participating postmenopausal women, which could have affected the HDL fraction. If menopause affected the transfer to HDL of lipids that constitute the core of the lipoprotein particle (namely, cholesteryl esters and triglycerides) or its surface layer (namely, phospholipids and free cholesterol), the size of the lipoprotein would also change. In this study, plasma HDL particles were of similar size in premenopausal and postmenopausal women, which corroborates the similar transfer of core and surface lipids to the lipoprotein that we documented. In addition its role in reverse cholesterol transport and cholesterol esterification, HDL has antioxidant, anti-inflammatory, antithrombotic, and vasodilation actions that may account for the antiatherogenic effect of the lipoprotein [20]. As the lipid transfer process is determinant of HDL composition and metabolism, it is possible that, if menopause affected the transfer of 1 or more lipids, the stability, reverse cholesterol transport operations, and protective functions of the lipoprotein could be compromised. In the methodological approach we used here, the rates of incorporation to HDL of all the major constituent lipids of the lipoprotein were simultaneously assessed by means of a standard donor, an artificial nanoemulsion. This is a practical and cost-effective tool to evaluate a crucial aspect of HDL metabolism, which reflects the compositional and functional status of the lipoprotein. A limitation of the study was its small sample size, however, and an investigation should be performed with much larger populations. In conclusion, when the effects of aging, hyperlipidemia, and obesity were excluded, menopause per se did not appear to primarily affect HDL levels, which are considered a major protective factor in women. Likewise, neither particle size in the HDL fraction nor lipid transfer to HDL (a fundamental aspect of the overall metabolism of this lipoprotein) was altered after menopause. It is possible, however, that HDL concentration and function may be indirectly affected when such alterations occur in women who experience weight gain and hyperlipidemia after postmenopause. Acknowledgements This study was supported by Fundação do Amparo à Pesquisa do Estado de São Paulo (FAPESP) and The Zerbini Foundation, both in São Paulo, Brazil. Dr Maranhão received a 1A Research Award from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brasilia, Brazil. References [1] Kannel WB, Levy D. Menopause, hormones, and cardiovascular vulnerability in women. Arch Intern Med 2004;164(5):479–81. [2] Kannel WB, McGee D, Gordon T. A general cardiovascular risk profile: the Framingham Study. Am J Cardiol 1976;38(1):46–51. [3] Matthews KA, Meilahn E, Kuller LH, Kelsey SF, Caggiula AW, Wing RR. Menopause and risk factors for coronary heart disease. N Engl J Med 1989;321(10):641–6. [4] Fukami K, Koike K, Hirota K, Yoshikawa H, Miyake A. Perimenopausal changes in serum lipids and lipoproteins: a 7-year longitudinal study. Maturitas 1995;22(3): 193–7. [5] Melo NR, Latrilha MC, Santos RD, Pompei LM, Maranhao RC. Effects in postmenopausal women of transdermal estrogen associated with progestin upon the removal from the plasma of a microemulsion that resembles low-density lipoprotein (LDL). Maturitas 2005;50(4):275–81. [6] Walsh BW, Schiff I, Rosner B. Effects of postmenopausal estrogen replacement on the concentrations and metabolism of plasma lipoproteins. N Engl J Med 1991;325(17): 1196–204. [7] Kim CJ, Kim TH, Ryu WS, Ryoo UH. Influence of menopause on high density lipoprotein-cholesterol and lipids. J Korean Med Sci 2000;1594):380–6.
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[8] Stevenson JC, Crook D, Godsland IF. Influence of age and menopause on serum lipids and lipoproteins in healthy women. Atherosclerosis 1993;98(1):83–90. [9] Matthews KA, Kuller LH, Sutton-Tyrrell K, Chang YF. Changes in cardiovascular risk factors during the perimenopause and postmenopause and carotid artery atherosclerosis in healthy women. Stroke 2001;32(5):1104–11. [10] Mercuro G, Zoncu S, Dragoni F. Gender differences in cardiovascular risk factors. Ital Heart J 2003;4(6):363–6. [11] Peters HW, Westendorp IC, Hak AE, Grobbee DE, Stehouwer CD, Hofman A, et al. Menopausal status and risk factors for cardiovascular disease. J Intern Med 1999;246(6):521–8. [12] Lima ES, Maranhao RC. Rapid, simple laser-light-scattering method for HDL particle sizing in whole plasma. Clin Chem 2004;50(6):1086–8. [13] Ginsburg GS, Small DM, Atkinson D. Microemulsions of phospholipids and cholesterol esters: protein-free models of low density lipoprotein. J Biol Chem 1982;257(14):8216–27. [14] Maranhao RC, Garicochea B, Silva EL, Dorlhiac-Llacer P, Cadena SM, Coelho IJ, et al. Plasma kinetics and biodistribution of a lipid emulsion resembling low-density lipoprotein in patients with acute leukemia. Cancer Res 1994;54(17):4660–6.
[15] Bainton D, Miller NE, Bolton CH, Yarnell JW, Sweetnam PM, Baker IA, et al. Plasma triglyceride and high density lipoprotein cholesterol as predictors of ischaemic heart disease in British men: The Caerphilly and Speedwell Collaborative Heart Disease Studies. Br Heart J 1992;68(1):60–6. [16] Wang M, Briggs MR. HDL: the metabolism, function, and therapeutic importance. Chem Rev 2004;104(1):119–37. [17] Norata GD, Pirillo A, Catapano AL. Modified HDL: biological and physiopathological consequences. Nutr Metab Cardiovasc Dis 2006;16(5):371–86. [18] Jia L, Long S, Fu M, Yan B, Tian Y, Xu Y, et al. Relationship between total cholesterol/ high-density lipoprotein cholesterol ratio, triglyceride/high-density lipoprotein cholesterol ratio, and high-density lipoprotein subclasses. Metabolism 2006;55 (5):1141–8. [19] Ushiroyama T, Sakuma K, Ikeda A, Ueki M. The HDL2/HDL3 ratio in menopause. Int J Gynecol Obstet 2005;88(3):303–8. [20] Ansell BJ, Watson KE, Fogelman AM, Navab M, Fonarow GC. High-density lipoprotein function: recent advances. J Am Coll Cardiol 2005;46(10):1792–8.
Contents lists available at ScienceDirect
International Journal of Gynecology and Obstetrics j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i j g o
CLINICAL ARTICLE
HDL concentration, lipid transfer to HDL, and HDL size in normolipidemic nonobese menopausal women Arícia H. Giribela a, Nilson R. Melo a, Maria C. Latrilha b, Edmundo C. Baracat a, Raul C. Maranhão b,c,⁎ a b c
Gynecological Department and Lipid Metabolism Laboratory, University of Sao Paulo, São Paulo, Brazil Heart Institute (INCOR) of the School of Medicine, University of Sao Paulo, São Paulo, Brazil Faculty of Pharmaceutical Sciences, University of Sao Paulo, São Paulo, Brazil
a r t i c l e
i n f o
Article history: Received 20 June 2008 Received in revised form 15 September 2008 Accepted 1 October 2008 Keywords: Aging Cholesterol Estrogen High-density lipoproteins Lipid transfer Menopause Triglycerides
a b s t r a c t Objective: To determine the impact of menopause on lipid transfer from donor lipoproteins to high-density lipoproteins (HDLs)—a process that is related to the protective function of HDL—and the size of HDL particles. Method: Plasma from 22 premenopausal and 18 postmenopausal nonobese, normolipidemic women paired for age (40–50 years) was incubated in an artificial nanoemulsion labeled with radioactive lipids. Then the HDL fraction was assessed for radioactivity; the percentage of radioactive lipids transferred from the nanoemulsion to HDL was determined; and the size of HDL particles was measured by laser light scattering. Results: There were no differences between the 2 groups in serum concentration of HDL cholesterol (61 ± 12 mg/dL vs 61 ± 14 mg/dL) or apolipoprotein A1 (1.5 ± 0.3 g/L vs 1.5 ± 0.2 g/L); lipid transfer to HDL; or size of HDL particles (8.8 ± 0.8 vs 9.0 ± 0.5 nm). Conclusion: Menopause was not found to affect HDL cholesterol plasma concentration, lipid transfer to HDL, or size of HDL particles in normolipidemic nonobese women. © 2008 International Federation of Gynecology and Obstetrics. Published by Elsevier Ireland Ltd. All rights reserved.
1. Introduction For those affected, coronary artery disease (CAD) typically develops 10 to 15 years later in women than in men [1]. As the plasma concentration of low-density lipoprotein cholesterol (LDL-C) is positively correlated with CAD, and the plasma concentration of highdensity lipoprotein cholesterol (HDL-C) is negatively correlated with CAD, plasma lipid status is one of the cardinal factors involved in atherogenesis [2]. In women, LDL-C and HDL-C levels are, respectively, lower and higher than in men, and lower HDL-C levels are more consistently predictive of the appearance of CAD symptoms [3]. Menopause is generally considered a turning point for the risk of CAD [3]. The delay in CAD development in women is attributed to hormonal protection before menopause, which, it is argued, is lost after ovarian hormone production ceases [1]. Fukami et al. [4] showed that the metabolic changes take place within the year preceding menopause. Most studies show that LDL-C concentration rises after menopause and Mathews et al. [3] observed that it rises sharply within 6 months of the cessation of menses. Decreasing estrogen levels are associated with a decrease in LDL-C removal from blood plasma, a condition that hormone treatment can revert [5,6].
In contrast, the effect of menopause on HDL-C is uncertain. There have been reports of increased [7], decreased [8,9], or sustained [8,9] levels; moreover, Mercuro et al. [10] reported that after menopause, concurrently with a gradual decrease in HDL-C concentration over 2 years, there was a rise in apolipoprotein A1 levels, which implies a possible change in HDL composition. Aging is the prominent factor in the appearance of CAD. In both sexes, the aging process results in a diminution of the removal of LDL-C from blood plasma, resulting in increased LDL-C levels. As the effect of age on HDL-C is as controversial as that of menopause, the concomitant—even synergistic—effects of aging and menopause on plasma lipids should be rigorously set apart [11]. By selecting premenopausal and postmenopausal women of the same age range, this cross-sectional study evaluated exclusively the impact of menopause on plasma lipid concentration, lipid transfer to HDL, and size of HDL particles. To avoid the interference of elevated serum levels of LDL-C and triglycerides on HDL metabolism, only women with serum cholesterol levels less than 200 mg/dL and triglyceride levels less than 150 mg/dL were included. The similar levels of those lipids in all participants are therefore due to the inclusion criteria. 2. Materials and methods
⁎ Corresponding author. Instituto do Coração (InCor) do Hospital das Clínicas da USP, Laboratório de Metabolismo de Lípides, Av. Dr. Enéas C. Aguiar 44, 1o Subsolo, CEP05403-000, São Paulo-SP, Brazil. Fax: +55 11 30695574. E-mail address: [email protected] (R.C. Maranhão).
A total of 22 consecutive premenopausal and 18 postmenopausal healthy women were enrolled at the Gynecological Department of Clinics Hospital of the University of São Paulo, where they were
0020-7292/$ – see front matter © 2008 International Federation of Gynecology and Obstetrics. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijgo.2008.10.001
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receiving periodic checkups. They were aged between 40 and 50 years and had a total cholesterol level less than 200 mg/dL, a LDL-C level less than 120 mg/dL, and a triglyceride level less than 150 mg/dL. Exclusion criteria were obesity, defined as a body mass index (BMI, calculated as weight in kilograms divided by the square of height in meters) greater than 30; habitual consumption of tobacco or alcohol; apparent or reported cardiovascular disease; diabetes and/or other metabolic disease; hypertension; cancer; pregnancy or breastfeeding; and use of contraceptives, hormone therapy, or antihyperlipidemic drugs. No participant had a history of any disease known to affect lipid metabolism. None was engaging in regular training exercises and all followed a typical local diet, without dietary restrictions. All had normal plasma levels of thyroid stimulating hormone, tri-iodothyronine, and free thyroxine. Menopausal status was defined by the presence or absence of amenorrhea and elevated plasma levels of follicle-stimulating hormone (FSH). The participants were considered postmenopausal if their last period occurred more than 1 year before study entry and their FSH plasma level was greater than 30 mUI/mL. They were considered premenopausal if they had regular cycles, no menopauserelated symptoms, and a FSH level less than 30 mUI/mL. Physical data of the participants are shown in Table 1. The study objective and design were explained to each participant, and all gave written informed consent. The study was approved by the Scientific and Ethics Committee of the University of São Paulo Medical School Hospital. Blood samples were collected by venipuncture after a 12-hour fast. Commercial enzymatic kits were used for the determination of plasma levels of glucose (Boehringer Mannheim, Penzberg, Germany), total cholesterol (Boehringer Mannheim), and triglycerides (Abbott Laboratories, Abbott Park, IL, USA). Levels of HDL-C were determined by the method used for total cholesterol after lipoprotein precipitation with magnesium phosphotungstate; levels of LDL-C were calculated by the Friedewald formula. Apolipoproteins A1 and B were determined by radial immunodiffusion (Boehringer Mannheim). As described previously [12], the size of HDL particles was measured by laser light scattering in the plasma supernatant after chemical precipitation of the lipoproteins containing apolipoprotein B. We used a ZetaPALS Zeta Potential analyzer (Brookhaven Intruments, Holtsville, NY, USA). The nanoemulsion contained a lipid mixture composed of 40 mg of cholesteryl oleate, 20 mg of egg phosphaditylcholine, 1 mg of triolein, and 0.5 mg of cholesterol (all from Sigma-Aldrich, St Louis, MO, USA), to which were added the radioactive lipids (Amersham International, Amersham, UK). It was obtained by the Ginsburg method [13]
Table 1 Physical characteristics, plasma glucose, lipids and apolipoproteins of the study subjects a Variable
Premenopausal group (n = 22)
Postmenopausal group (n = 18)
P value
Age, y Weight, kg Height, m BMI Abdominal circumference, cm Fasting glucose, mg/dL Cholesterol, mg/dL Total HDL-C LDL-C Triglycerides, mg/dL Apolipoprotein A1, g/L Apolipoprotein B, g/L
45 ± 3 61.5 ± 8.2 1.60 ± 0.10 24.9 ± 2.6 81.0 ± 10.4
48 ± 3 62.8 ± 8.3 1.60 ± 0.10 24.5 ± 3.1 84.1 ± 9.0
b 0.01 NS NS NS NS
86 ± 9.1
86 ± 6.8
NS
191 ± 27 61 ± 12 103 ± 35 103 ± 50 1.5 ± 0.33 0.9 ± 0.22
178 ± 24 61± 14 100 ± 24 80 ± 44 1.5 ± 0.22 0.8 ± 0.20
NS NS NS NS NS NS
Abbreviations: BMI, body mass index (calculated as weight in kilograms divided by the square of height in meters); HDL-C, high-density lipoprotein cholesterol; LDL-C, lowdensity lipoprotein cholesterol; NS, not significant. a Values are given as mean ± SD unless otherwise indicated.
Table 2 HDL size and lipid transfer from the nanoemulsion to HDLa Variable Diameter, nm Lipid transfer, % b Free cholesterol Phospholipids Esterified cholesterol Triglycerides
Premenopausal group (n = 22)
Postmenopausal group (n = 18)
P value
9.0 ± 0.5
8.8 ± 0.8
NS
5.6 ± 0.9 17.8 ± 2.1 3.1 ± 1.7 3.6 ± 0.7
5.3 ± 0.8 18.8 ± 1.9 2.7 ± 0.8 3.6 ± 0.9
NS NS NS NS
a
Values are given as mean ± SD. The value indicated is the percentage of the radioactivity of each lipid component in the nanoemulsion that was transferred to the plasma HDL fraction after a 60-minute incubation. The ability of the HDL fraction to receive lipids from LDL, such as free cholesterol, cholesteryl esters, phospholipids, and triglycerides, was measured. This process depends on transfer proteins, which mediate the transfer of lipids from one lipoprotein class to another, and also on the composition and structure of the recipient HDL. Additionally, we evaluated the HDL particle size.
b
modified by Maranhão et al. [14], where lipids are emulsified in an aqueous medium by prolonged ultrasonic irradiation, and the crude emulsion then ultracentrifugated in a 2-step process, with density adjustment by addition of potassium bromide. Two sets of the nanoemulsion were prepared, one labeled with 3H-cholesteryl oleate and 14C-cholesterol and the other with 14C-phosphatidylcholine and 3H-triolein. We then assessed the transfer to plasma HDL of the radioactive lipids present in the nanoemulsion. Plasma was obtained from centrifugating, at 2500 rpm and 4 °C for 15-minutes, blood samples collected into glass tubes containing 0.15% Na2 EDTA. Tubes containing 0.2 mL of plasma and 0.05 mL of a nanoemulsion labeled with either 3 H-cholesteryl oleate and 14C-cholesterol or 14C-phosphatidylcholine and 3H-triolein were placed in a shaking bath and incubated for 60 minutes at 37 °C. Then, 0.25 mL of a solution containing 0.02% of dextran sulfate (molecular weight, 50 000) and 0.3 mol/L of magnesium chloride was added to the tube and vortexed for 30 seconds. Samples were centrifuged at 3000 rpm for 10 minutes and 0.25 mL of the obtained supernatant, which contained the plasma HDL fraction, were transferred to vials containing a scintillation solution, vigorously shaken, and left to stand for at least 45 minutes. Radioactivity was determined using a Packard 1660 TR liquid scintillation counter (Packard BioScience, Meriden, Connecticut, USA). The transfer of the nanoemulsion lipids to HDL was evaluated as percentage of the radioactivity of a given lipid formerly in the nanoemulsion, and found in the plasma HDL fraction after the 60minute incubation period. The statistical differences between the 2 groups were evaluated using the Graf Pad Instat software, version 3.0 (Graf Pad Software, San Diego, CA, USA), by the t test because the groups showed normal distribution. Data were expressed as mean ± standard deviation. P b 0.05 was considered significant in all analyses. 3. Results The participants were slightly older in the postmenopausal than in the premenopausal group (Table 1), but BMI and plasma glucose concentration were similar in the 2 groups. There were no differences between the 2 groups in plasma concentrations of total cholesterol, LDL-C, HDL-C, triglycerides, apolipoprotein A1, or apolipoprotein B. The diameter of HDL particles was similar in the 2 groups (Table 2). Table 2 also shows the results of the assay by which the transfer of free and esterified cholesterol, triglycerides, and phospholipids from the nanoemulsion to HDL was evaluated. There were no differences between the 2 groups regarding the percentage of acceptance by HDL of any of the 4 types of lipids, whether the values were obtained from the entire HDL fraction or from the normalized HDL-C concentration.
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No correlation was found between the lipid values obtained in this study and BMI, abdominal circumference, or mean group age. In the postmenopausal group the lipid values did not correlate with time elapsed since menopause. 4. Discussion In this study, only women with serum fasting cholesterol less than 200 mg/dL and triglycerides less than 150 mg/dL were included. The similar levels of those lipids in the premenopausal and the postmenopausal group are therefore due to the inclusion criteria and cannot be interpreted as a lack of effect of menopause on plasma concentration of LDL-C or on triglyceride-rich lipoproteins. The see-saw effect of triglyceride levels on HDL is well known, by which HDL levels decrease as triglyceride levels increase, and reciprocally [15]. Since the metabolisms of the different classes of plasma lipoprotein are intertwined, by including only women with total cholesterol and triglyceride less than the cut-off values for risk of CAD, the effects on HDL of alterations of lipoprotein from other classes— such as LDL or very-low-density lipoproteins (VLDL)—were avoided. Therefore, because of the exclusion criteria used to distinguish the primary effects of menopause on HDL metabolism, the interpretation of our results cannot be extended to women who gain weight or become hyperlipidemic during or following menopause. Because of the effects of age on lipid plasma levels and the regulation of lipoprotein metabolism, another criterion in the selection of the participants was age. The participants were about 3 years older in the postmenopausal group, but it did not influence the results. Despite the limitations inherent in cross-sectional studies, the fact that there were no differences between the 2 groups in any of the parameters measured suggests that the hormonal changes occurring during menopause did not influence those parameters. One of the HDL functions in plasma lipid regulation is reverse transport of cholesterol. In reverse transport, HDL removes cholesterol from the peripheral tissues and transports it to the liver, where it is excreted in the bile. Alternatively HDL may transfer cholesterol to lipoproteins from other classes, but the fate of the transferred cholesterol will be the same. The transfer proteins therefore play a major role in the cholesterol removal process. Cholesteryl ester transfer protein (CETP) and phospholipid transfer protein (PLTP) are the major proteins involved in those lipid shifts. The former specifically mediates the transfer of triglycerides from VLDL to LDL and HDL, as well as the transfer of cholesterol esters from LDL and HDL to VLDL. It is associated with HDL in the plasma. As for PLTP, it is partially associated with HDL and mediates the transfer of phospholipids from VLDL to HDL, as well as the reciprocal exchange of phospholipids between VLDL and HDL. Although lipid transfers between lipoproteins of different classes are bidirectional, they frequently result in lipid enrichment or depletion in a given class [16]. It has been increasingly perceived that not only HDL plasma concentration but also the qualitative and functional aspects of HDL are important for its antiatherogenic actions, as is exemplified by HDL containing the Milano mutation of apolipoprotein A1. Even with low levels of circulating HDL, individuals carrying this apolipoprotein A1 variant are protected from cardiovascular events. Similarly, individuals with high levels of HDL that are prone to modification could be less protected than others with lesser levels of HDL that are more resistant to modification [17]. The exchange of lipids to and from HDL particles is crucial to reverse cholesterol transport, and it clearly requires a coordinated metabolic regulation of the HDL subclasses. In recent years, it has been considered that changes in the distribution of HDL subclasses of smaller or larger particles were more closely correlated with atherosclerosis than low plasma levels of HDL-C. Some studies have reported that small-size HDL particles are associated with an increased risk of coronary heart disease, whereas
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large-size HDL particles are associated with a decreased risk [18]. The lack of effect of menopause on particle size in the HDL fraction in the present study suggests that menopause does not change the HDL subclass profile. In this respect, Ushiroyama et al. [19] and Stevenson et al. [8] reported that there were fewer HDL2 (larger) particles and more HDL3 (smaller) particles in the plasma of postmenopausal women, compared with premenopausal women. However, this finding can be attributed to increased triglyceride levels in the participating postmenopausal women, which could have affected the HDL fraction. If menopause affected the transfer to HDL of lipids that constitute the core of the lipoprotein particle (namely, cholesteryl esters and triglycerides) or its surface layer (namely, phospholipids and free cholesterol), the size of the lipoprotein would also change. In this study, plasma HDL particles were of similar size in premenopausal and postmenopausal women, which corroborates the similar transfer of core and surface lipids to the lipoprotein that we documented. In addition its role in reverse cholesterol transport and cholesterol esterification, HDL has antioxidant, anti-inflammatory, antithrombotic, and vasodilation actions that may account for the antiatherogenic effect of the lipoprotein [20]. As the lipid transfer process is determinant of HDL composition and metabolism, it is possible that, if menopause affected the transfer of 1 or more lipids, the stability, reverse cholesterol transport operations, and protective functions of the lipoprotein could be compromised. In the methodological approach we used here, the rates of incorporation to HDL of all the major constituent lipids of the lipoprotein were simultaneously assessed by means of a standard donor, an artificial nanoemulsion. This is a practical and cost-effective tool to evaluate a crucial aspect of HDL metabolism, which reflects the compositional and functional status of the lipoprotein. A limitation of the study was its small sample size, however, and an investigation should be performed with much larger populations. In conclusion, when the effects of aging, hyperlipidemia, and obesity were excluded, menopause per se did not appear to primarily affect HDL levels, which are considered a major protective factor in women. Likewise, neither particle size in the HDL fraction nor lipid transfer to HDL (a fundamental aspect of the overall metabolism of this lipoprotein) was altered after menopause. It is possible, however, that HDL concentration and function may be indirectly affected when such alterations occur in women who experience weight gain and hyperlipidemia after postmenopause. Acknowledgements This study was supported by Fundação do Amparo à Pesquisa do Estado de São Paulo (FAPESP) and The Zerbini Foundation, both in São Paulo, Brazil. Dr Maranhão received a 1A Research Award from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brasilia, Brazil. References [1] Kannel WB, Levy D. Menopause, hormones, and cardiovascular vulnerability in women. Arch Intern Med 2004;164(5):479–81. [2] Kannel WB, McGee D, Gordon T. A general cardiovascular risk profile: the Framingham Study. Am J Cardiol 1976;38(1):46–51. [3] Matthews KA, Meilahn E, Kuller LH, Kelsey SF, Caggiula AW, Wing RR. Menopause and risk factors for coronary heart disease. N Engl J Med 1989;321(10):641–6. [4] Fukami K, Koike K, Hirota K, Yoshikawa H, Miyake A. Perimenopausal changes in serum lipids and lipoproteins: a 7-year longitudinal study. Maturitas 1995;22(3): 193–7. [5] Melo NR, Latrilha MC, Santos RD, Pompei LM, Maranhao RC. Effects in postmenopausal women of transdermal estrogen associated with progestin upon the removal from the plasma of a microemulsion that resembles low-density lipoprotein (LDL). Maturitas 2005;50(4):275–81. [6] Walsh BW, Schiff I, Rosner B. Effects of postmenopausal estrogen replacement on the concentrations and metabolism of plasma lipoproteins. N Engl J Med 1991;325(17): 1196–204. [7] Kim CJ, Kim TH, Ryu WS, Ryoo UH. Influence of menopause on high density lipoprotein-cholesterol and lipids. J Korean Med Sci 2000;1594):380–6.
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[8] Stevenson JC, Crook D, Godsland IF. Influence of age and menopause on serum lipids and lipoproteins in healthy women. Atherosclerosis 1993;98(1):83–90. [9] Matthews KA, Kuller LH, Sutton-Tyrrell K, Chang YF. Changes in cardiovascular risk factors during the perimenopause and postmenopause and carotid artery atherosclerosis in healthy women. Stroke 2001;32(5):1104–11. [10] Mercuro G, Zoncu S, Dragoni F. Gender differences in cardiovascular risk factors. Ital Heart J 2003;4(6):363–6. [11] Peters HW, Westendorp IC, Hak AE, Grobbee DE, Stehouwer CD, Hofman A, et al. Menopausal status and risk factors for cardiovascular disease. J Intern Med 1999;246(6):521–8. [12] Lima ES, Maranhao RC. Rapid, simple laser-light-scattering method for HDL particle sizing in whole plasma. Clin Chem 2004;50(6):1086–8. [13] Ginsburg GS, Small DM, Atkinson D. Microemulsions of phospholipids and cholesterol esters: protein-free models of low density lipoprotein. J Biol Chem 1982;257(14):8216–27. [14] Maranhao RC, Garicochea B, Silva EL, Dorlhiac-Llacer P, Cadena SM, Coelho IJ, et al. Plasma kinetics and biodistribution of a lipid emulsion resembling low-density lipoprotein in patients with acute leukemia. Cancer Res 1994;54(17):4660–6.
[15] Bainton D, Miller NE, Bolton CH, Yarnell JW, Sweetnam PM, Baker IA, et al. Plasma triglyceride and high density lipoprotein cholesterol as predictors of ischaemic heart disease in British men: The Caerphilly and Speedwell Collaborative Heart Disease Studies. Br Heart J 1992;68(1):60–6. [16] Wang M, Briggs MR. HDL: the metabolism, function, and therapeutic importance. Chem Rev 2004;104(1):119–37. [17] Norata GD, Pirillo A, Catapano AL. Modified HDL: biological and physiopathological consequences. Nutr Metab Cardiovasc Dis 2006;16(5):371–86. [18] Jia L, Long S, Fu M, Yan B, Tian Y, Xu Y, et al. Relationship between total cholesterol/ high-density lipoprotein cholesterol ratio, triglyceride/high-density lipoprotein cholesterol ratio, and high-density lipoprotein subclasses. Metabolism 2006;55 (5):1141–8. [19] Ushiroyama T, Sakuma K, Ikeda A, Ueki M. The HDL2/HDL3 ratio in menopause. Int J Gynecol Obstet 2005;88(3):303–8. [20] Ansell BJ, Watson KE, Fogelman AM, Navab M, Fonarow GC. High-density lipoprotein function: recent advances. J Am Coll Cardiol 2005;46(10):1792–8.