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HDL3 ratio in menopause
International Journal of Gynecology and Obstetrics (2005) 88, 303 — 308
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CLINICAL ARTICLE
The HDL2/HDL3 ratio in menopause T. Ushiroyama*, K. Sakuma, A. Ikeda, M. Ueki Department of Obstetrics and Gynecology, Osaka Medical College, 2-7 Daigaku-machi, Takatsuki, Osaka 569-8686, Japan Received 15 September 2004; received in revised form 13 December 2004; accepted 22 December 2004
KEYWORDS Menopause; HDL2/3 ratio; Arteriosclerosis; Hyperlipidemia; Triglyceride
Abstract Objectives: The influence of the menopause on the HDL2/HDL3 ratio was assessed in association with hypertriglyceridemia. Methods: Fasting blood samples were collected from 607 patients. Commercially available enzymatic methods were used for determination of TG, and total HDL-C. HDL2 and HDL3 were measured by ultracentrifugation. Results: The HDL2/HDL3 ratio had a strong negative correlation with TG (r= 0.272, Pb0.0001 and r=-0.314, Pb0.0001) in both pre- and postmenopausal women. No significant differences were observed in HDL2, HDL3, and HDL2/HDL3 ratio between pre- and postmenopausal women without hypertriglyceridemia. Postmenopausal women had a significantly higher HDL2/HDL3 ratio than premenopausal women with hypertriglyceridemia. Conclusions: These results indicate that menopausal status not only increases plasma LDL-cholesterol and triglyceride levels, but also increases the HDL2/HDL3 ratio when associated with elevation of plasma triglyceride levels. These changes may increase the risk for CHD due to enlargement of the lipid pool. D 2005 International Federation of Gynecology and Obstetrics. Published by Elsevier Ireland Ltd. All rights reserved.
1. Introduction An elevated level of serum cholesterol, known as hypercholesterolemia, is undoubtedly associated with an increased risk of premature coronary
* Corresponding author. Tel.: +81 72 683 1221; fax: +81 72 681 3723. E-mail address: [email protected] (T. Ushiroyama).
atherosclerosis. The Framingham Heart Study [1] and the British Regional Heart Study [2] supported the belief that plasma levels of cholesterol correlate with risk of cardiovascular events. Estrogen deficiency occurring at the menopause, whether spontaneous or induced surgically, is accompanied by increased serum cholesterol and triglyceride levels [3]. We note that 27.2% of perimenopausal and 22.0% of postmenopausal women in Japan had elevated low-density lipoprotein cholesterol (LDLC) and triglyceride (TG) levels [3]. The relationship
0020-7292/$ - see front matter D 2005 International Federation of Gynecology and Obstetrics. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijgo.2004.12.017
304 between cardiovascular events and hypertriglyceridemia, especially TG-rich lipoprotein metabolic changes, has recently been the focus of considerable research. The dramatic changes in lipoprotein metabolism that occur around the menopause include an increase in the concentration of LDL cholesterol and remnant-like particle lipoprotein cholesterol (RLP-C) [3,4]. Epidemiological studies indicate that elevated high-density lipoprotein cholesterol (HDL-C) concentrations are associated with a lower incidence of cardiovascular disease [5] and that this antiatherogenic effect may be due to the physiologic function of HDL-C in reverse cholesterol transport. Low levels of HDL-C (b40 mg/dl), as observed in patients with hypertriglyceridemia, are considered to be a risk factor for cardiovascular disease [6]. The therapeutic correction of hypertriglyceridemia with pharmacological agents such as bezafibrate is associated with a beneficial increase in plasma HDL-C concentrations together with a decrease of triglyceride content and coagulation activity [7]. It has been generally accepted that HDL cholesterol concentration decreases with the menopause [8]. However, a few recent reports have demonstrated no difference between periand postmenopausal women, although LDL-C levels in perimenopausal women were lower than in postmenopausal women [9,10]. At present, no consensus of opinion has been reached on the influence of the menopause on plasma total HDL-C levels. The composition of HDL-C, i.e., the ratio of HDL2/HDL3, is a modulating factor in the lipoprotein’s structure, and modifications of the lipoprotein (especially of the HDL3 subfraction) are known to induce changes in function, for example, the capacity of HDL3 to efflux intracellular cholesterol ester [11], the mechanism by which excess cholesterol is removed from peripheral tissues for catabolism by the liver. The risk of coronary heart disease is lower in women than in men, but increases in women after the menopause. Some of the gender-, age-, and menopause-related differences in coronary heart disease risk may relate to differences in lipoprotein subspecies. In a smaller study, it was reported that postmenopausal status was associated with significantly higher values of LDL-C, lower levels of HDL-C, and smaller HDL particle size [12]. The aim of the present study is to examine the influence of the menopause on HDL particle size and the HDL2/HDL3 ratio, and changes of these measurements associated with elevation of plasma TG level.
T. Ushiroyama et al.
2. Materials and methods 2.1. Materials A total of 607 patients (age: 50.5F9.6 years; 37— 73 years) from the middle-aged outpatients clinic at the Osaka Medical College Hospital were screened for plasma lipid levels. All of these women were in good health, and all gave informed consent to participate in the study. None of the patients had a history of cardiovascular disease or hormone therapy, and none had undergone treatment with a lipid-lowering drug prior to the study. Menopausal status was determined by the absence of menses for at least 6 months and bilateral oophorectomy before entering the study and was determined by high plasma levels of gonadotropins and low plasma levels of estradiol (FSHN30 mIU/ml, LHN25 mIU/ml, estradiolb10 pg/ml).
2.2. Laboratory methods After an overnight fast, venous blood samples were collected for analyses of serum total cholesterol (T-CHO), TG, HDL-C, RLP-C, HDL2 (large HDL), and HDL3 (small HDL) concentrations. Commercially available enzymatic methods were used for determination of T-CHO, TG, and HDL-C. HDL-C fractions were obtained with the dextran sulfate—magnesium chloride precipitation techniques developed by Talameh et al. [13] and assayed with a commercial kit (HDL-C2 Daiichi, Tokyo, Japan) from Daiichi Chemical Pharmacy. Tokyo, Japan. LDL-C levels were obtained from calculations using the formula of Friedewald [14]. HDL 2 and 3 were isolated and measured by ultracentrifugation according to the recommendations of Schumaker and Puppione [15]. Plasma RLP-C was determined by immunoabsorption using the commercial RLP-C JIMRO kit. Plasma gonadotropin (LH and FSH) and estradiol levels were measured using a commercially available enzyme immunoassay kit (bioMerieux-Vitek, Ltd., Tokyo, Japan).
2.3. Statistical analysis All values are expressed as meanFS.D. Correlations were evaluated using simple regression analysis. The statistical significance of the differences between each value was determined using Wilcoxon’s signed-ranks test. All differences were considered significant at Pb0.05.
The HDL2/HDL3 ratio in menopause
305
HDL2, HDL3 (mg/dl)
Overall, there were no major differences in the baseline data for height, weight, and daily habits between pre- (n=251) and postmenopausal women (n=356). There were significant differences in BMI, plasma LDL-cholesterol ( P=0.017), and triglyceride ( P=0.015) levels between pre- and postmenopausal women. Differences were also observed in endocrine background levels (plasma FSH, LH, estradiol, and estrone; Pb0.0001) due to the menopause. The mean age of the two groups differed by approximately 10 years. Fig. 1 shows the results of simple regression analysis between plasma triglyceride level and the HDL2/HDL3 ratio. The HDL2/HDL3 ratio had a strong negative correlation with TG (r= 0.272, Pb0.0001 and r= 0.314, Pb0.0001) in both preand postmenopausal women. Although postmenopausal women had significantly higher plasma LDLcholesterol ( P=0.017) and triglyceride ( P=0.015) than premenopausal women, we observed no significant differences in plasma HDL2, HDL3, and HDL2/HDL3 ratio between the two groups (Fig. 2). In order to analyze the effect of the menopause on plasma HDL2 and HDL3 concentrations without
Premenopause (n=251)
Premenopause (n=251) Postmenopause (n=356)
50
6 5
40
4
30
3
20
2
10
1
0
HDL2/HDL3
60
3. Results
0 HDL2
HDL3
HDL2/HDL3
Figure 2 The comparison of plasma HDL2 and HDL3 concentrations, and HDL2/HDL3 ratio between in preand postmenopausal women.
the influence of age, the lipid parameters of women within the same perimenopausal age band (50—52 years) were compared (Fig. 3). Lipid parameters were compared between pre- (n=130) and postmenopausal women (n=165) aged 50—52 years. Plasma HDL2 ( P=0.043), HDL3 concentration ( P=0.0006), and HDL2/HDL3 ratio ( P=0.012) showed significant differences, while plasma LDLcholesterol and triglyceride concentrations showed
7
R=0.272 P<0.0001
6 5
HDL2 / HDL3
4 3 2 1 0 0
50
100 150 200 250 300 350 400 mg/dl
TG
Postmenopause (n=356)
7 R=0.314 P<0.0001
6 5
HDL2 / HDL3
4 3 2 1 0 0
100
200
300
TG
400
500
600
mg/dl
Figure 1 The correlation of plasma triglyceride levels with HDL2/HDL3 ratio. (Upper: Premenopause, Lower: Postmenopause).
306
T. Ushiroyama et al.
Postmenopause (n=165) P=0.043
60
6
300 50 40
200
30
150
P=0.0006
100
20
50
10 0
0 LDL-cholesterol Triglyceride
HDL2
HDL3
5 4 3 2
HDL2/HDL3
P=0.012
250
HDL2, HDL3 (mg/dl)
LDL-Cholesterol, Triglyceride (mg/dl)
Premenopause (n=130)
1 0
HDL2/HDL3
Figure 3 The comparison of plasma LDL cholesterol, triglyceride, HDL2, HDL3 concentrations, and HDL2/HDL3 ratio between in pre- and postmenopausal women 50—52 years of age around menopause after adjusting for age.
HDL3 ratio (3.11F1.2 and 3.14F1.1) whose plasma TG level was below 150 mg/dl (mean 87.6F27.9 mg/dl and 83.7F29.0 mg/dl, respectively;Fig. 4, lower). However, in women with plasma TG levels above 150 mg/dl, the postmenopausal HDL2/HDL3 Cases with high plasma triglyceride level (n=67) 60
Premenopause (n=31)
Premenopause (n=56)
50
Postmenopause (n=89)
6 5
P=0.041
40
4
30
3
20
2
10
1
HDL2, HDL3 (mg/dl)
60
HDL2/HDL3
HDL2, HDL3 (mg/dl)
Cases with high plasma triglyceride level (n=145) 50
P=0.048
P=0.025
5
40
4
30
3
20
2
10
1 0
0
HDL2 0
6
Postmenopause (n=36)
HDL2/HDL3
no significant differences between pre- and postmenopausal women after adjusting for age. There were no significant differences between the pre- and postmenopausal women in plasma HDL2 (41.2F12.5 mg/dl and 42.0F12.0 mg/dl, respectively) and HDL3 concentrations (14.0F3.0 mg/dl and 14.2F4.2 mg/dl, respectively) or HDL2/
HDL3
HDL2/HDL3
0
HDL2
HDL3
Cases with low plasma triglyceride level (n=228)
HDL2/HDL3
P=0.014
6
50
Postmenopause (n=267)
5
40
4
30
3
20
2
10
1
0
0
50
P=0.0016
HDL3
HDL2/HDL3
Figure 4 The comparison of plasma HDL2 and HDL3 concentrations, and HDL2/HDL3 ratio between in preand postmenopausal women based on different plasma triglyceride range. Upper: TriglycerideR150 mg/dl, Lower: Triglycerideb150 mg/dl.
5 4
40 30
3
P=0.0007
20
2
10
1 0
0
HDL2
6
Postmenopause (n=129)
HDL2/HDL3
Premenopause (n=195)
HDL2, HDL3 (mg/dl)
60
Premenopause (n=99)
60
HDL2/HDL3
HDL2, HDL3 (mg/dl)
Cases with low plasma triglyceride level (n=462)
HDL2
HDL3
HDL2/HDL3
Figure 5 The comparison of plasma HDL2 and HDL3 concentrations, and HDL2/HDL3 ratio between in preand postmenopausal women 50—52 years of age around menopause after adjusting for age on the basis of different plasma triglyceride range. Upper: TriglycerideR150 mg/dl, Lower: Triglycerideb150 mg/dl.
The HDL2/HDL3 ratio in menopause ratio (2.85F1.2) was significantly higher than the premenopausal ratio (2.45F1.1, P=0.041; Fig. 4, upper). The differences in mean values of lipoproteins between pre-and postmenopausal women aged 50 to 52 years are shown in Fig. 5. We observed significant differences in plasma HDL2 ( P=0.014), plasma HDL3 ( P=0.0007), and HDL2/ HDL3 ratio ( P=0.0016) between the pre- (n=99) and postmenopausal (n=129) women whose plasma TG level was below 150 mg/dl after adjusting for age (Fig. 5, lower). Postmenopausal women showed a significantly lower HDL ( 11.7%) and HDL2/HDL3 ratio ( 16.8%) and a higher HDL3 (9.3%) than premenopausal women. However, in women with plasma TG levels above 150 mg/dl, the change from premenopausal to postmenopausal status was associated with an 11.7% increase in HDL2 ( P=0.048) and a 13.4% increase in HDL2/HDL3 ratio ( P=0.025; Fig. 5, upper).
4. Discussion Recent studies [16,17] have shown that HDL3 mediated or correlated with cholesterol efflux and elevation of cyclic-AMP, and concurrently enhanced cholesterol efflux and binding of HDL3 in human skin fibroblast in vitro. Panzenboeck et al. [18] reported that HDL3 was a better promotor of cholesterol efflux than lipid-free apo A-I. In a clinical report, Marcil [19] revealed that familial HDL deficiency was also characterized by reduced HDL3 and apo A-I-mediated cellular cholesterol efflux. The present study sought to assess the influence of the menopause on qualitative changes of HDL by examining the ratio of large HDL (HDL2) to small HDL (HDL3) in the triglyceride metabolic system. One of the main functions of HDL is to effect the removal of surplus cholesterol from cells [20]. The effects seen with the change from premenopausal to postmenopausal status were a decrease in HDL2 and an increase in HDL3 with a reduction of HDL2/HDL3 ratio after standardization for age. Stevenson et al. reported increases in LDL cholesterol and triglyceride with a striking reduction in HDL2 concentration with the menopause [21]. They concluded that the menopause is associated with potentially adverse changes in lipids and lipoproteins, independent of any effects of ageing. Another longitudinal study has reported findings similar to their cross-sectional observations [22]. Our data demonstrated that menopausal status did not alter HDL particle size in women within
307 normal triglyceride levels. However, menopausal status increased the HDL2/HDL3 ratio in women with elevated triglyceride levels. In other words, when plasma triglyceride levels were high (over 150 mg/dl), menopausal status created a metabolic condition with a relatively high concentration of HDL2 and a relatively low concentration of HDL3. This study demonstrated that menopausal status clearly raised plasma HDL2 and the HDL2/HDL3 ratio in women with hyperlipidemia when we analyzed subjects aged 50 to 52 years. This indicates that the menopause may be a factor which alters the quality of HDL in women with abnormal triglyceride metabolism. Estrogens modulate lipid metabolism and the increased risk of atherosclerosis in postmenopausal women is at least partly due to reduction of estrogen production after the menopause. The menopause results not only in the reduction of estrogen production but also in the elevation of BMI, and a change of lipid metabolism which should be independent of estrogen level. The menopause itself, or unknown age-related factors, may contribute to these changes. In the management of hyperglyceridemia in postmenopausal women, emphasis should be placed on increasing intracellular cholesterol effluxing capacity by decreasing plasma TG and RLP-C levels, and by increasing the HDL2/HDL3 ratio, as well as suppression of LDL production, and increasing the uptake of LDL by LDL receptors in the liver. As expected, a positive effect of hormone replacement therapy (HRT) was seen on the atherogenic lipid profile [23]. Estrogen stimulates an increase in LDL receptors in the liver, and increases HDL with a concomitant increase in Apo A due to reduced activity of hepatic lipase, the enzyme that catabolizes HDL [24]. However, HRT is also known to increase hepatic production of very low density lipoprotein, and therefore an increase in triglyceride was expected [24]. The clinical importance of HRT on the treatment of dyslipidemia is still controversial [25]. Medical intervention is suggested to recover the plasma TG level to physiological levels, and to reduce HDL2/HDL3 ratio as countermeasures for postmenopausal hypertriglyceridemia and decreased HDL cholesterol. This is potentially a very interesting area for future investigation. There is a major debate about the costeffectiveness of screening for dyslipidemia in women with hypertriglyceridemia. Monitoring HDL particle size in the postmenopausal period, even if there is no change in total HDL concentration, may reduce the risk of developing coronary heart disease.
308
References [1] Friedman GD, Kannel WB, Dawber TR. Comparison of prevalence, case history and incidence data in assessing the potency of risk factors in coronary heart disease. Am J Epidemiol 1966;83:366 – 78. [2] Shaper AG, Pocock SJ, Walker M. Risk factors for ischemic heart disease: the prospective phase of the British Regional Heart Study. J Epidemiol Community Health 1985;39:197 – 209. [3] Ushiroyama T, Okamoto Y, Sugimoto O. Plasma lipid and lipoprotein levels in perimenopausal women—clinical research in 1198 Japanese women. Acta Obstet Gynecol Scand 1993;72:428 – 33. [4] Ushiroyama T, Ikeda A, Higashiyama T, Orino I, Ueki M. Effects of bezafibrate on serum lipid concentrations in hypertriglyceridemic perimenopausal patients and on—with effects on serum remnant like particles (RLP-C) metabolism and endocrinologic changes. J Jpn Menopause Soc 1996;4: 240 – 5. [5] Manninen V, Tenkanen L, Koskinen P. Joint effects of serum triglycerides and LDL cholesterol and HDL cholesterol concentrations on coronary heart disease risk in the Helsinki Heart Study. Circulation 1992;85:37 – 45. [6] Eisenberg S, Gavish D, Oschry Y, Fainaru M, Deckenbaum RJ. Abnormalities in very low, low, and high density lipoproteins in hypertriglyceridemia. Reversal towards normal with bezafibrate treatment. J Clin Invest 1984;74:470 – 82. [7] Ushiroyama T, Ikeda A, Higashio S, Ueki M. Coagulofibrinolysis assessment of effects of bezafibrate on hypertriglyceridemia in postmenopausal women. Blood Coagul Fibrinolysis 2000;11:709 – 14. [8] Middleberg RP, Spector TD, Awaminathan R. Genetic and environmental influence on lipids, lipoproteins, and apolipoproteins: effects of menopause. Arterioscler Thromb Vasc Biol 2002;22:1142 – 7. [9] Kim CJ, Kim TH, Ryu WS. Influence of menopause on high density lipoprotein-cholesterol and lipids. J Korean Med Sci 2000;15:380 – 6. [10] Peters HW, Westendorp IC, Hak AE, Grobbee DE, Stehouwer A, Hofman A, et al. Menopausal status and risk factors for cardiovascular disease. J Intern Med 1999;246:521 – 8. [11] Va Ville AE, Sola R, Balanya J, Turner PR, Masana L. In vitro oxidized HDL is recognized by the scavenger receptor of macrophages: implications for its protective role in vivo. Atherosclerosis 1994;106:179 – 89. [12] Li Z, McNamara JR, Fruchart JC, Luc G, Bard JM, Ordovas JM, et al. Effects of gender and menopausal status on plasma lipoprotein subspecies and particle sizes. J Lipid Res 1996;37:1886 – 96.
T. Ushiroyama et al. [13] Talameh Y, Wei R, Naito H. Measurements of total HDL, HDL2, HDL3 by dextran sulfate—MgCl2 precipitation technique in human serum. Clin Chim Acta 1986;158:33 – 9. [14] Friedewald WT, Levy RI, Frederikson DS. Estimation of the concentration of low-density lipoprotein cholesterol in plasma without use of the preparative ultracentrifuge. Clin Chem 1972;18:499 – 502. [15] Schumaker UN, Puppione DL. Sequential flotation ultracentrifugation. Methods Enzymol 1986;128:155 – 70. [16] Langer C, Huang Y, Cullen P, Wiesenhutter B, Mahley RW, Assmann G, et al. Endogenous apolipoprotein E modulates cholesterol efflux and cholesteryl ester hydrolysis mediated by high-density lipoprotein-3 and lipid-free apolipoproteins in mouse peritoneal macrophages. J Mol Med 2000;78:217 – 27. [17] Senault C, Betoulle D, Luc G, Hauw P, Rigard D, Fumeron F. Beneficial effects of a moderate consumption of red wine on cellular cholesterol efflux in young men. Nutr Metab Cardiovasc Dis 2000;10:63 – 9. [18] Panzenboeck U, Balazs Z, Sovic A, Hrzenjak A, LevakFrank S, Wintersperger A, et al. ABCA1 and scavenger receptor class B, type I, are modulators of reverse sterol transport at an in vitro blood—brain barrier constituted of porcine brain capillary endothelial cells. J Biol Chem 2000;277:42781 – 9. [19] Marcil M, Yu L, Krimbou L, Boucher B, Oram JF, Cohn JS, et al. Cellular cholesterol transport and efflux in fibroblasts are abnormal in subjects with familial HDL deficiency. Arterioscler Thromb Vasc Biol 1999;19:150 – 69. [20] Oram JF. Effects of high density lipoprotein subfractions on cholesterol homeostasis in human fibroblasts and arterial smooth muscle cells. Arteriosclerosis 1983;3: 420 – 32. [21] Stevenson JC, Crook D, Godsland IF. Influence of age and menopause on serum lipids and lipoproteins in healthy women. Atherosclerosis 1993;98:83 – 90. [22] Jensen J, Nilas L, Christiansen C. Influence of menopause on serum lipids and lipoproteins. Maturitas 1990;12:321 – 6. [23] Bjarnason NH, Byrjalsen I, Hassager C, Haarbo J, Christiansen C. Low dose of estradiol in combination with gestogene to prevent early postmenopausal bone loss. Am J Obstet Gynecol 2000;183:550 – 60. [24] Christiansen C. Sex steroids and the cardiovascular system. Osteoporos Int 1997;7:S8 – 11 [Suppl.]. [25] Sanada M, Nakagawa H, Kodama I, Ohama K. Three-year study of oestrogen alone versus combined with progestin in postmenopausal women with and without hypercholesterolemia. Metabolism 2000;49:784 – 9.
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CLINICAL ARTICLE
The HDL2/HDL3 ratio in menopause T. Ushiroyama*, K. Sakuma, A. Ikeda, M. Ueki Department of Obstetrics and Gynecology, Osaka Medical College, 2-7 Daigaku-machi, Takatsuki, Osaka 569-8686, Japan Received 15 September 2004; received in revised form 13 December 2004; accepted 22 December 2004
KEYWORDS Menopause; HDL2/3 ratio; Arteriosclerosis; Hyperlipidemia; Triglyceride
Abstract Objectives: The influence of the menopause on the HDL2/HDL3 ratio was assessed in association with hypertriglyceridemia. Methods: Fasting blood samples were collected from 607 patients. Commercially available enzymatic methods were used for determination of TG, and total HDL-C. HDL2 and HDL3 were measured by ultracentrifugation. Results: The HDL2/HDL3 ratio had a strong negative correlation with TG (r= 0.272, Pb0.0001 and r=-0.314, Pb0.0001) in both pre- and postmenopausal women. No significant differences were observed in HDL2, HDL3, and HDL2/HDL3 ratio between pre- and postmenopausal women without hypertriglyceridemia. Postmenopausal women had a significantly higher HDL2/HDL3 ratio than premenopausal women with hypertriglyceridemia. Conclusions: These results indicate that menopausal status not only increases plasma LDL-cholesterol and triglyceride levels, but also increases the HDL2/HDL3 ratio when associated with elevation of plasma triglyceride levels. These changes may increase the risk for CHD due to enlargement of the lipid pool. D 2005 International Federation of Gynecology and Obstetrics. Published by Elsevier Ireland Ltd. All rights reserved.
1. Introduction An elevated level of serum cholesterol, known as hypercholesterolemia, is undoubtedly associated with an increased risk of premature coronary
* Corresponding author. Tel.: +81 72 683 1221; fax: +81 72 681 3723. E-mail address: [email protected] (T. Ushiroyama).
atherosclerosis. The Framingham Heart Study [1] and the British Regional Heart Study [2] supported the belief that plasma levels of cholesterol correlate with risk of cardiovascular events. Estrogen deficiency occurring at the menopause, whether spontaneous or induced surgically, is accompanied by increased serum cholesterol and triglyceride levels [3]. We note that 27.2% of perimenopausal and 22.0% of postmenopausal women in Japan had elevated low-density lipoprotein cholesterol (LDLC) and triglyceride (TG) levels [3]. The relationship
0020-7292/$ - see front matter D 2005 International Federation of Gynecology and Obstetrics. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijgo.2004.12.017
304 between cardiovascular events and hypertriglyceridemia, especially TG-rich lipoprotein metabolic changes, has recently been the focus of considerable research. The dramatic changes in lipoprotein metabolism that occur around the menopause include an increase in the concentration of LDL cholesterol and remnant-like particle lipoprotein cholesterol (RLP-C) [3,4]. Epidemiological studies indicate that elevated high-density lipoprotein cholesterol (HDL-C) concentrations are associated with a lower incidence of cardiovascular disease [5] and that this antiatherogenic effect may be due to the physiologic function of HDL-C in reverse cholesterol transport. Low levels of HDL-C (b40 mg/dl), as observed in patients with hypertriglyceridemia, are considered to be a risk factor for cardiovascular disease [6]. The therapeutic correction of hypertriglyceridemia with pharmacological agents such as bezafibrate is associated with a beneficial increase in plasma HDL-C concentrations together with a decrease of triglyceride content and coagulation activity [7]. It has been generally accepted that HDL cholesterol concentration decreases with the menopause [8]. However, a few recent reports have demonstrated no difference between periand postmenopausal women, although LDL-C levels in perimenopausal women were lower than in postmenopausal women [9,10]. At present, no consensus of opinion has been reached on the influence of the menopause on plasma total HDL-C levels. The composition of HDL-C, i.e., the ratio of HDL2/HDL3, is a modulating factor in the lipoprotein’s structure, and modifications of the lipoprotein (especially of the HDL3 subfraction) are known to induce changes in function, for example, the capacity of HDL3 to efflux intracellular cholesterol ester [11], the mechanism by which excess cholesterol is removed from peripheral tissues for catabolism by the liver. The risk of coronary heart disease is lower in women than in men, but increases in women after the menopause. Some of the gender-, age-, and menopause-related differences in coronary heart disease risk may relate to differences in lipoprotein subspecies. In a smaller study, it was reported that postmenopausal status was associated with significantly higher values of LDL-C, lower levels of HDL-C, and smaller HDL particle size [12]. The aim of the present study is to examine the influence of the menopause on HDL particle size and the HDL2/HDL3 ratio, and changes of these measurements associated with elevation of plasma TG level.
T. Ushiroyama et al.
2. Materials and methods 2.1. Materials A total of 607 patients (age: 50.5F9.6 years; 37— 73 years) from the middle-aged outpatients clinic at the Osaka Medical College Hospital were screened for plasma lipid levels. All of these women were in good health, and all gave informed consent to participate in the study. None of the patients had a history of cardiovascular disease or hormone therapy, and none had undergone treatment with a lipid-lowering drug prior to the study. Menopausal status was determined by the absence of menses for at least 6 months and bilateral oophorectomy before entering the study and was determined by high plasma levels of gonadotropins and low plasma levels of estradiol (FSHN30 mIU/ml, LHN25 mIU/ml, estradiolb10 pg/ml).
2.2. Laboratory methods After an overnight fast, venous blood samples were collected for analyses of serum total cholesterol (T-CHO), TG, HDL-C, RLP-C, HDL2 (large HDL), and HDL3 (small HDL) concentrations. Commercially available enzymatic methods were used for determination of T-CHO, TG, and HDL-C. HDL-C fractions were obtained with the dextran sulfate—magnesium chloride precipitation techniques developed by Talameh et al. [13] and assayed with a commercial kit (HDL-C2 Daiichi, Tokyo, Japan) from Daiichi Chemical Pharmacy. Tokyo, Japan. LDL-C levels were obtained from calculations using the formula of Friedewald [14]. HDL 2 and 3 were isolated and measured by ultracentrifugation according to the recommendations of Schumaker and Puppione [15]. Plasma RLP-C was determined by immunoabsorption using the commercial RLP-C JIMRO kit. Plasma gonadotropin (LH and FSH) and estradiol levels were measured using a commercially available enzyme immunoassay kit (bioMerieux-Vitek, Ltd., Tokyo, Japan).
2.3. Statistical analysis All values are expressed as meanFS.D. Correlations were evaluated using simple regression analysis. The statistical significance of the differences between each value was determined using Wilcoxon’s signed-ranks test. All differences were considered significant at Pb0.05.
The HDL2/HDL3 ratio in menopause
305
HDL2, HDL3 (mg/dl)
Overall, there were no major differences in the baseline data for height, weight, and daily habits between pre- (n=251) and postmenopausal women (n=356). There were significant differences in BMI, plasma LDL-cholesterol ( P=0.017), and triglyceride ( P=0.015) levels between pre- and postmenopausal women. Differences were also observed in endocrine background levels (plasma FSH, LH, estradiol, and estrone; Pb0.0001) due to the menopause. The mean age of the two groups differed by approximately 10 years. Fig. 1 shows the results of simple regression analysis between plasma triglyceride level and the HDL2/HDL3 ratio. The HDL2/HDL3 ratio had a strong negative correlation with TG (r= 0.272, Pb0.0001 and r= 0.314, Pb0.0001) in both preand postmenopausal women. Although postmenopausal women had significantly higher plasma LDLcholesterol ( P=0.017) and triglyceride ( P=0.015) than premenopausal women, we observed no significant differences in plasma HDL2, HDL3, and HDL2/HDL3 ratio between the two groups (Fig. 2). In order to analyze the effect of the menopause on plasma HDL2 and HDL3 concentrations without
Premenopause (n=251)
Premenopause (n=251) Postmenopause (n=356)
50
6 5
40
4
30
3
20
2
10
1
0
HDL2/HDL3
60
3. Results
0 HDL2
HDL3
HDL2/HDL3
Figure 2 The comparison of plasma HDL2 and HDL3 concentrations, and HDL2/HDL3 ratio between in preand postmenopausal women.
the influence of age, the lipid parameters of women within the same perimenopausal age band (50—52 years) were compared (Fig. 3). Lipid parameters were compared between pre- (n=130) and postmenopausal women (n=165) aged 50—52 years. Plasma HDL2 ( P=0.043), HDL3 concentration ( P=0.0006), and HDL2/HDL3 ratio ( P=0.012) showed significant differences, while plasma LDLcholesterol and triglyceride concentrations showed
7
R=0.272 P<0.0001
6 5
HDL2 / HDL3
4 3 2 1 0 0
50
100 150 200 250 300 350 400 mg/dl
TG
Postmenopause (n=356)
7 R=0.314 P<0.0001
6 5
HDL2 / HDL3
4 3 2 1 0 0
100
200
300
TG
400
500
600
mg/dl
Figure 1 The correlation of plasma triglyceride levels with HDL2/HDL3 ratio. (Upper: Premenopause, Lower: Postmenopause).
306
T. Ushiroyama et al.
Postmenopause (n=165) P=0.043
60
6
300 50 40
200
30
150
P=0.0006
100
20
50
10 0
0 LDL-cholesterol Triglyceride
HDL2
HDL3
5 4 3 2
HDL2/HDL3
P=0.012
250
HDL2, HDL3 (mg/dl)
LDL-Cholesterol, Triglyceride (mg/dl)
Premenopause (n=130)
1 0
HDL2/HDL3
Figure 3 The comparison of plasma LDL cholesterol, triglyceride, HDL2, HDL3 concentrations, and HDL2/HDL3 ratio between in pre- and postmenopausal women 50—52 years of age around menopause after adjusting for age.
HDL3 ratio (3.11F1.2 and 3.14F1.1) whose plasma TG level was below 150 mg/dl (mean 87.6F27.9 mg/dl and 83.7F29.0 mg/dl, respectively;Fig. 4, lower). However, in women with plasma TG levels above 150 mg/dl, the postmenopausal HDL2/HDL3 Cases with high plasma triglyceride level (n=67) 60
Premenopause (n=31)
Premenopause (n=56)
50
Postmenopause (n=89)
6 5
P=0.041
40
4
30
3
20
2
10
1
HDL2, HDL3 (mg/dl)
60
HDL2/HDL3
HDL2, HDL3 (mg/dl)
Cases with high plasma triglyceride level (n=145) 50
P=0.048
P=0.025
5
40
4
30
3
20
2
10
1 0
0
HDL2 0
6
Postmenopause (n=36)
HDL2/HDL3
no significant differences between pre- and postmenopausal women after adjusting for age. There were no significant differences between the pre- and postmenopausal women in plasma HDL2 (41.2F12.5 mg/dl and 42.0F12.0 mg/dl, respectively) and HDL3 concentrations (14.0F3.0 mg/dl and 14.2F4.2 mg/dl, respectively) or HDL2/
HDL3
HDL2/HDL3
0
HDL2
HDL3
Cases with low plasma triglyceride level (n=228)
HDL2/HDL3
P=0.014
6
50
Postmenopause (n=267)
5
40
4
30
3
20
2
10
1
0
0
50
P=0.0016
HDL3
HDL2/HDL3
Figure 4 The comparison of plasma HDL2 and HDL3 concentrations, and HDL2/HDL3 ratio between in preand postmenopausal women based on different plasma triglyceride range. Upper: TriglycerideR150 mg/dl, Lower: Triglycerideb150 mg/dl.
5 4
40 30
3
P=0.0007
20
2
10
1 0
0
HDL2
6
Postmenopause (n=129)
HDL2/HDL3
Premenopause (n=195)
HDL2, HDL3 (mg/dl)
60
Premenopause (n=99)
60
HDL2/HDL3
HDL2, HDL3 (mg/dl)
Cases with low plasma triglyceride level (n=462)
HDL2
HDL3
HDL2/HDL3
Figure 5 The comparison of plasma HDL2 and HDL3 concentrations, and HDL2/HDL3 ratio between in preand postmenopausal women 50—52 years of age around menopause after adjusting for age on the basis of different plasma triglyceride range. Upper: TriglycerideR150 mg/dl, Lower: Triglycerideb150 mg/dl.
The HDL2/HDL3 ratio in menopause ratio (2.85F1.2) was significantly higher than the premenopausal ratio (2.45F1.1, P=0.041; Fig. 4, upper). The differences in mean values of lipoproteins between pre-and postmenopausal women aged 50 to 52 years are shown in Fig. 5. We observed significant differences in plasma HDL2 ( P=0.014), plasma HDL3 ( P=0.0007), and HDL2/ HDL3 ratio ( P=0.0016) between the pre- (n=99) and postmenopausal (n=129) women whose plasma TG level was below 150 mg/dl after adjusting for age (Fig. 5, lower). Postmenopausal women showed a significantly lower HDL ( 11.7%) and HDL2/HDL3 ratio ( 16.8%) and a higher HDL3 (9.3%) than premenopausal women. However, in women with plasma TG levels above 150 mg/dl, the change from premenopausal to postmenopausal status was associated with an 11.7% increase in HDL2 ( P=0.048) and a 13.4% increase in HDL2/HDL3 ratio ( P=0.025; Fig. 5, upper).
4. Discussion Recent studies [16,17] have shown that HDL3 mediated or correlated with cholesterol efflux and elevation of cyclic-AMP, and concurrently enhanced cholesterol efflux and binding of HDL3 in human skin fibroblast in vitro. Panzenboeck et al. [18] reported that HDL3 was a better promotor of cholesterol efflux than lipid-free apo A-I. In a clinical report, Marcil [19] revealed that familial HDL deficiency was also characterized by reduced HDL3 and apo A-I-mediated cellular cholesterol efflux. The present study sought to assess the influence of the menopause on qualitative changes of HDL by examining the ratio of large HDL (HDL2) to small HDL (HDL3) in the triglyceride metabolic system. One of the main functions of HDL is to effect the removal of surplus cholesterol from cells [20]. The effects seen with the change from premenopausal to postmenopausal status were a decrease in HDL2 and an increase in HDL3 with a reduction of HDL2/HDL3 ratio after standardization for age. Stevenson et al. reported increases in LDL cholesterol and triglyceride with a striking reduction in HDL2 concentration with the menopause [21]. They concluded that the menopause is associated with potentially adverse changes in lipids and lipoproteins, independent of any effects of ageing. Another longitudinal study has reported findings similar to their cross-sectional observations [22]. Our data demonstrated that menopausal status did not alter HDL particle size in women within
307 normal triglyceride levels. However, menopausal status increased the HDL2/HDL3 ratio in women with elevated triglyceride levels. In other words, when plasma triglyceride levels were high (over 150 mg/dl), menopausal status created a metabolic condition with a relatively high concentration of HDL2 and a relatively low concentration of HDL3. This study demonstrated that menopausal status clearly raised plasma HDL2 and the HDL2/HDL3 ratio in women with hyperlipidemia when we analyzed subjects aged 50 to 52 years. This indicates that the menopause may be a factor which alters the quality of HDL in women with abnormal triglyceride metabolism. Estrogens modulate lipid metabolism and the increased risk of atherosclerosis in postmenopausal women is at least partly due to reduction of estrogen production after the menopause. The menopause results not only in the reduction of estrogen production but also in the elevation of BMI, and a change of lipid metabolism which should be independent of estrogen level. The menopause itself, or unknown age-related factors, may contribute to these changes. In the management of hyperglyceridemia in postmenopausal women, emphasis should be placed on increasing intracellular cholesterol effluxing capacity by decreasing plasma TG and RLP-C levels, and by increasing the HDL2/HDL3 ratio, as well as suppression of LDL production, and increasing the uptake of LDL by LDL receptors in the liver. As expected, a positive effect of hormone replacement therapy (HRT) was seen on the atherogenic lipid profile [23]. Estrogen stimulates an increase in LDL receptors in the liver, and increases HDL with a concomitant increase in Apo A due to reduced activity of hepatic lipase, the enzyme that catabolizes HDL [24]. However, HRT is also known to increase hepatic production of very low density lipoprotein, and therefore an increase in triglyceride was expected [24]. The clinical importance of HRT on the treatment of dyslipidemia is still controversial [25]. Medical intervention is suggested to recover the plasma TG level to physiological levels, and to reduce HDL2/HDL3 ratio as countermeasures for postmenopausal hypertriglyceridemia and decreased HDL cholesterol. This is potentially a very interesting area for future investigation. There is a major debate about the costeffectiveness of screening for dyslipidemia in women with hypertriglyceridemia. Monitoring HDL particle size in the postmenopausal period, even if there is no change in total HDL concentration, may reduce the risk of developing coronary heart disease.
308
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