Influence of menopause on blood cholesterol levels in women: the role of body composition, fat distribution and hormonal milieu

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1 Journal of Internal Medicine 1997; 241: Influence of menopause on blood cholesterol levels in women: the role of body composition, fat distribution and hormonal milieu R. PASQUALIa, F. CASIMIRRIa, G. PASCALc, O. TORTELLIc, A. M. MORSELLI LABATEa, D. BERTAZZOa, V. VICENNATIa, A. GADDIb, AND THE VIRGILIO MENOPAUSE HEALTH GROUP* From the aendocrinology Section, Department of Internal Medicine and Gastroenterology, and bgiancarlo Descovich Atherosclerosis Centre, St Orsola-Malpighi Hospital, University of Bologna, Bologna; and cdivision of Medicine, Carlo Poma Hospital, Mantua; Italy Abstract. Pasquali R, Casimirri F, Pascal G, Tortelli O, Mari A, Labate M, Bertazzo D, Vicennati V, Gaddi A, and the Virgilio Menopause Health Group (St Orsola- Malpighi Hospital, University of Bologna, Bologna; Carlo Poma Hospital, Mantua; Italy). Influence of menopause on blood cholesterol levels in women: the role of body composition, fat distribution and hormonal milieu. J Intern Med 1997; 241: Objectives. In this study we investigated the relationships between blood lipids and menopausal status. Setting and subjects. All data were obtained from the first cross-sectional examination of the Virgilio Menopause Health Project in a large cohort of middle-aged women in pre, peri-, and postmenopausal age. The data refer to 426 women without metabolic or endocrine diseases, relevant hepatic, renal and cardiovascular abnormalities, none were dieting or taking medications. Main outcome measures. A precoded questionnaire including full clinical history, socio-economic and personal information, habitual diet, physical activity, drug use and smoking habits, careful recording of gynaecological events and family history for disease was completed. Several anthropometric parameters and the bioelectrical impedance analysis was used to measure free fatty mass. Blood samples for hormones and biochemistry were also obtained. Results. There were no significant differences on body mass index, fatty mass, free fatty mass and parameters of body fat distribution between the three groups. Again, there were no differences in smoking habits, dietary intake or indices of physical activity amongst the groups. There was a significant increase from pre to postmenopause of LH and FSH and a decrease of oestradiol and testosterone, whereas no difference was found in sex hormone-binding globulin. Age-adjusted values of glucose, triglycerides and high density lipoprotein (HDL-) cholesterol were similar in all groups, whereas postmenopausal women had significantly higher values of total and low density lipoprotein (LDL-) cholesterol. On the contrary, there was a significant fall in insulin levels passing from pre to postmenopause. In multiple regression models, total and LDL-cholesterol correlated positively with body mass index, waist-to-hip ratio and age, and negatively with free fatty mass and oestradiol blood levels. Conclusions. These results are consistent with the hypothesis that menopausal status may have a significant and independent effect in determining increased total and LDL-cholesterol concentrations in postmenopausal women. Keywords: body composition, body fat distribution, menopause, oestrogens. *Participants in the Virgilio Menopause Health Study: S. Cantobelli, B. Anconetani (Institute of Clinical Medicine 1, St Orsola Hospital, Bologna); R. Chattat, M. Ercolani (Institute of Psychology, University of Bologna); P. Spina, A. Bertuzzi, A. Bondavalli, D. Belloni (Carlo Poma Hospital, Mantua); A. Battesini, D. Biagi, C. Carreta, C. Falavigna, M. Galavotti, E. Mondini, P. Pizzi, S. Venco (General Practitioners, Virgilio, Mantua) Blackwell Science Ltd 195

2 196 RENATO PASQUALI et al. Introduction There are very few studies on the effects of menopause and related hormonal patterns on lipids and lipoproteins. Despite this it is commonly believed that both normal and surgical menopause are associated with altered lipoprotein changes as well as with increased vascular risk [1]. In two prospective studies women who became postmenopausal showed a slight but significant reduction in high density lipoprotein (HDL-) cholesterol and an increase in triglycerides and low density lipoprotein (LDL-) cholesterol as compared with premenopausal women [2, 3]. However, Kuller et al. [4] found no significant changes in LDL-cholesterol concentrations in women passing from premenopausal to postmenopausal status, whereas they observed that the HDL2 fraction and apoprotein A-1 were decreased, particularly in those with very low blood-oestrogen concentrations. In a cross-sectional study Gambrel & Jensen found a significant increase in total and LDL cholesterol without any significant variation in triglycerides or HDL-cholesterol concentrations [5]. Despite the extensive research on the effects of oestrogens and progestagens on lipid and lipoprotein metabolism [6, 7] it is not yet clear whether changes in sex steroid concentrations are related to changes in lipid concentrations associated with the menopause status. Moreover, there are very few data on the effects of body weight and fat distributions on lipid concentrations in middle-aged women ranging from premenopause to postmenopause. Therefore, in this study we investigated the relationship between the menopausal status and related hormonal variation with plasma lipid concentrations, taking into account the effects of age, body weight, fatty and free-fatty mass, body fat distribution, dietary habits and physical activity. All data presented here were obtained from the first crosssectional examination of a longitudinal survey performed in Virgilio, Mantova (Italy) in a large cohort of women in pre-, peri- and postmenopausal status [8]. Material and methods Subjects and protocol of the study The subject population evaluated in this study included 426 women participating in the Virgilio Menopause Health Project, a longitudinal study whose general characteristics have been reported previously [8]. This sample represents 74% of the original cohort participating in first cross-sectional survey. Based on clinical history and laboratory data, all women having diabetes, thyroid disease or relevant cardiovascular, renal, hepatic or systemic diseases, or taking medications known to interfere with cholesterol synthesis or metabolism (including oestrogens and or progestagens) were excluded. All women gave their informed consent to the study and all procedures followed in this study were in accordance with the Second Helsinki Declaration. Women were evaluated in the morning after a 12-h fast. All evaluations were performed by the same well-trained investigator (O.T.) for the duration of the study. Blood samples were obtained randomly throughout the menstrual cycle, and the date of the previous bleeding was registered for an appropriate definition of the phase of the cycle in which the samples were taken. After blood samples had been drawn, they were maintained at C and centrifuged within 60 min. They were immediately frozen at 20 C and moved into a 76 C freezer within 1 or 2 days. In the evaluation the investigator used a precoded questionnaire [8] including a full clinical history, socio-economic and personal information, habitual or current drug use, smoking habits, careful recording of principal gynaecological events, a complete history of weight variations during the subject s life, family history of first-degree relatives diseases such as diabetes, obesity (women were asked to give information on their parents body weight and size), hypertension, hypercholesterolaemia and myocardial infarction, according to general criteria previously used [9]. Dietary habits were investigated by using the 24-h food recall interview technique according to Beaton [10] using bidimensional models of food portions, as reported elsewhere [11]. Physical activity was investigated by using a self-administered questionnaire as proposed by Baecke et al. [12] which makes it possible to distinguish three meaningful factors, scored from 1 to 5, such as (i) physical activity at work (work index), (ii) sport during leisure time (sport index), and (iii) physical activity during leisure time, excluding sport (leisure time index). Anthropometric measurements were obtained whilst women wore underwear. Height (to the nearest cm) and weight (to the nearest 0.5 kg) were

3 CHOLESTEROL LEVELS AND MENOPAUSAL STATUS 197 recorded. The body mass index (BMI) was calculated by dividing body weight, in kg, by height, in m. The following skinfolds were measured in duplicate with a Harpender calliper (British Indicator Ltd, UK) (and values were read when they started to stabilize): biceps, triceps, and subscapular. Circumferences were measured in the standing position at the following levels [13]: waist 1: smallest between lateral costal margin and iliac crest; waist 2: midway between lower rib margin and the iliac crest; waist 3: at the level of the iliac crest, passing along the umbilicus; hip: widest circumference over the great trochanters; thigh: around the right thigh at the level of the gluteal fold; breast: widest circumference over the breast; arm: midway between the elbow and the humerus scapular joint. The following ratio circumference (waist-to-hip; W1HR, W2HR, W3HR) values were calculated. It was possible to perform bioelectrical impedance analysis (BIA) measurement only in 295 women (69.2% of the whole sample). BIA was performed using a tetrapolar-phase-sensitive impedance plethysmograph (50 khz) BIA 101S, Akern RJL Systems Inc. (Florence, Italy). BIA is a safe and validated method of estimating body fluids and free fatty masses (FFM) in humans [14]. Measurements were made whilst the subjects lay comfortably on a bed. All procedures were carried out according to the manufacturers instructions. Fatty mass (FM) was calculated by subtracting FFM from body weight. Definition of the menopausal status Definition of the menopausal status was performed in accordance with widely used criteria [15]. Women in amenorrhoea for at least 12 months, having oestradiol levels lower than 150 pmol L and FSH levels higher than 15 IU L, and women who had had bilateral ovariectomy and hysterectomy or were amenorrhoeic were included in the postmenopausal group (n 196). In this group, years after menopause ranged from 1 to 26, with a mean ( SD) of years. Those presenting normal menses or who reported at least 10 menstrual cycles in the antecedent year, and without climacteric-related symptoms (such as hot flushes, bleeding irregularities and fluctuation in mood) were included in the premenopausal group (n 133). Finally, women with the occurrence of at least one cycle in the previous 6-month period and or presenting hot flushes and or other symptoms related to the climacteric status were included in the peri-menopausal group (n 97). Hormones and biochemistry All hormones were determined in duplicate on serum samples stored at 76 C until assayed, as previously reported [8]. Blood lipid and glucose concentrations were determined immediately after blood samples had been drawn. Glucose was determined by the glucose-oxidase method. Total cholesterol and triglyceride levels were determined in plasma samples by enzymatic methods using reagents obtained from Biochemia Boehringer Robin (Mannheim, Germany). The HDL-cholesterol levels were measured after precipitation with MgCl 6H O (0.05 mmol L ) and phosphotungstic acid (14 mmol L ) with reagents purchased from Behring (Marburg, Germany). Lowdensity lipoprotein (LDL) cholesterol levels were calculated according to the formula of Friedewald et al. [16] Statistics Results are reported as mean SD. Data that did not fit the normal distribution, as assessed by the Kolmogorov Smirnov test [17], were adequately normalized using polynomial and or logarithmic transformations. The comparisons between pairs of groups were analysed by means of ANOVA (age) and ANCOVA, adjusting for age. Moreover the ANCOVA trend (linear contrast) was applied to evaluate the progressive effect of the menopausal status, passing from premenopause to peri- and postmenopause [18]. Stepwise multiple regression analyses [18], considering total LDL- and HDL-cholesterol as dependent variables, were also performed. Cigarette smoking habits and family history data were compared by using the chi-squared test. Results General characteristics, anthropometry and body composition Mean age values in the three groups (Table 1) were significantly (P 0.001) and progressively higher,

4 198 RENATO PASQUALI et al. Table 1 Anthropometric variables (mean SD) in pre-, peri-, and postmenopausal women Menopausal status ANCOVA Premenopause Perimenopause Postmenopause trend* Variables (n 133) (n 97) (n 196) (P-value) Age (years) a a,b Body weight (kg) BMI (kg m ) (130) W1HR a NS (194) W2HR a,b NS (194) W3HR a,b NS (194) FFM (kg) b (118) (86) (161) FM (kg) (118) (86) (161) Numbers in parentheses refer to the number of observations when different from the total number of women in each group. Symbols reported on the right of each column refer to the comparison between each group and the other two groups (ANCOVA), after adjusting for age (a P 0.01 for peri- and post- versus premenopause; b P 0.01 for post- versus perimenopause). * Refers to the progressive effects of the menopausal status evaluated by the ANCOVA trend, after adjusting for age values; indicates significant increasing decreasing trend, passing from pre-, peri-, to postmenopause. FFM, and FM, mean free fatty mass, and fatty mass, respectively. Table 2 Sex hormone and sex-hormone binding globulin blood concentrations (m SD) in pre-, peri-, and postmenopausal women Menopausal status ANCOVA Premenopause Perimenopause Postmenopause trend* Variables (n 133) (n 97) (n 196) (P-value) LH (IU L ) a a,b (114) (88) (168) FSH (IU L ) a a,b (114) (89) (168) Testosterone (nmol L ) a,b (113) (89) (167) Oestradiol (pmol L ) a a,b (114) (89) (166) SHBG (nmol L ) NS (116) (90) (178) Numbers in parentheses refer to the number of observations when different from the total number of women in each group. Symbols reported in each column refer to the comparison between each group and the others (ANCOVA), after adjusting for age: a: P 0.01 for post- versus premenopause; b: P 0.01 for post- versus perimenopause. * Refers to the progressive effects of the menopausal status evaluated by the ANCOVA trend, after adjusting for age values; indicates significant increasing decreasing trend, passing from pre-, peri-, to postmenopause. according to the menopausal status. All anthropometric parameters were significantly (P 0.05) and positively correlated with age. However, despite there being no observed significant differences in body weight, BMI, fatty mass and free fatty mass between the three groups, trend analysis indicated that menopausal status had a significant negative effect on body weight, fat mass, and free fatty mass, with respect to that expected on the basis of their relationship with age values. Although all waist-to-hip and waist-to-thigh ratios increased from pre- to peri- and postmenopausal women, the differences were not statistically significant after adjusting for age values.

5 CHOLESTEROL LEVELS AND MENOPAUSAL STATUS 199 Table 3 Dietary habits, physical activity and smoking habits (mean SD) in pre-, peri-, and postmenopausal women Menopausal status ANCOVA Premenopause Perimenopause Postmenopause trend* Variables (n 133) (n 97) (n 196) (P-value) Diet Energy (kj) b NS Proteins (g) NS Fats (g) NS Carbohydrates (g) b NS Fibres (g) NS Alcohol (g) NS Physical activity Work index a a NS (131) (97) (190) Leisure time index NS (130) (96) (190) Sport index NS (130) (96) (188) Total activity NS (128) (96) (184) Cigarette smoking status NS** Current smokers Non smokers Cigarettes day NS Numbers in parentheses refer to the number of observations when different from the total number of women in each group. Symbols reported in each column refer to the comparison between each group and the others (ANCOVA), after adjusting for age (a P 0.05 for peri- and post- versus premenopause; b P 0.01 for post- versus perimenopause. * Refers to the progressive effects of the menopausal status evaluated by the ANCOVA trend, after adjusting for age values; indicates significant increasing decreasing trend, passing from pre-, peri-, to postmenopause. ** Statistics: chi-squared test. Hormones and SHBG (Table 2) There was a significant and progressive increase in LH and FSH and decrease in oestradiol from premenopause to postmenopause. It is unlikely that these results were dependent, in some way, on the fact that in women with regular menses blood samples had been drawn randomly throughout the menstrual cycle. In fact, they were obtained, on average, on day 14.1 (median 13.8) of the cycle, and approximately 61% of the women were examined in the follicular phase. When data obtained from all other women examined in the luteal phase or at random in those presenting oligomenorrhoea were excluded, no significantly different values in gonadotropin and oestradiol were observed. There was also a significant difference in testosterone concentrations between the groups, as postmenopausal women had significantly lower values than pre- and perimenopausal women. However, there was no significant difference in the SHBG blood levels. Diet, physical activity and smoking habits (Table 3) Mean energy intake in the postmenopausal group was slightly but significantly (P 0.01) lower in comparison with the perimenopausal group. Accordingly, mean carbohydrate intake was significantly (P 0.01) lower in the postmenopausal versus perimenopausal groups. No significant difference was found in protein, lipid, fibre and alcohol intake. All groups had similar values of physical activity expressed by either sport, leisure time or total indices. Compared to the premenopausal

6 200 RENATO PASQUALI et al. Table 4 Insulin, glucose and lipid blood concentrations (mean SD) in pre-, peri-, and postmenopausal women Menopausal status ANCOVA Premenopause Perimenopause Postmenopause trend* Variables (n 133) (n 97) (n 196) (P-value) Insulin a (pmol L ) (114) (89) (166) Glucose NS (mmol L ) (129) Triglycerides a NS (mmol L ) (128) (195) Total cholesterol , b,c (mmol.l) (129) (195) HDL-cholesterol NS (mmol L ) (126) (94) (190) LDL-cholesterol b,c (mmol L ) (125) (94) (190) Numbers in parentheses refer to the number of observations when different from the total number of women in each group. Symbols reported in each column refer to the comparison between each group and the others (ANCOVA), after adjusting for age (a: P 0.05 and b: P 0.01 for post- versus premenopause; c: P 0.01 for post- versus perimenopause. * Refers to the progressive effects of the menopausal status evaluated by the ANCOVA trend, after adjusting for age values; indicates significant increasing decreasing trend, passing from pre-, peri-, to postmenopause. women, however, peri- and postmenopausal women had significantly higher values of work index (P 0.05). There were also no significant differences in smoking habits amongst the three groups. Insulin, glucose and lipids (Table 4) There were no differences in glucose and HDLcholesterol levels amongst the three groups. Although the ANCOVA between the three groups was not significant, comparison between pre- and postmenopausal women showed that triglyceride concentrations were significantly higher in post- versus premenopausal women. Moreover, there was a slight, but progressive decrease of insulin concentrations particularly in the postmenopausal group (P 0.05 vs. premenopause). On the contrary, postmenopausal women showed significantly higher total and LDLcholesterol concentrations in comparison with the other groups. Menopausal status and family history for diseases In the whole sample of women it was possible to obtain information on the family history in 323 women for diabetes, in 321 women for hypertension, in 322 women for myocardial infarction and hypercholesterolaemia, and in 320 women for obesity. There were no significant differences between the groups in the familial prevalence of diabetes, obesity and hypertension. On the contrary, postmenopausal women reported lower familiar prevalence of hypercholesterolaemia (17 192; 8.9%) and myocardial infarction (31 192; 16.1%) in comparison to premenopausal (21 130; 19.2%) and perimenopausal women (25 96; 26.0%), respectively. Interrelations between serum cholesterol levels, menopausal status and body composition (Table 5) In stepwise multiple regression models serum total or LDL-cholesterol levels were used as dependent variables, whereas age, anthropometric indices, energy and carbohydrate intake, the cumulative index of physical activity, insulin, glucose and their molar ratio, hormones (oestrogens and gonadotropins) and the menopausal status were included as independent variables. When the menopausal status was considered in the analysis, total cholesterol concentrations correlated significantly and positively with W1HR, BMI and the menopausal status itself, and negatively with FFM values. Alternatively, when the menopausal status was not considered, total cholesterol levels maintained similar correlations with W1HR, BMI, and FFM, and the positive contribution of the menopausal status was replaced by a significant positive correlation with age and a negative correlation with oestradiol values. Similar results

7 CHOLESTEROL LEVELS AND MENOPAUSAL STATUS 201 Table 5. Results of the stepwise multiple regression analysis applied in the whole sample of women participating in the Virgilio- Menopause-Health Study. Total (regression A) and LDL-cholesterol (regression B) were alternatively used as dependent variables. In regression A, the Menopausal status variable was alternatively considered (A1) or not (A2) as an independent variable. Variables Dependent Independent Multiple (r-value) P t-value P-value A1 Total cholesterol W1HR BMI FFM Menopausal status A2 Total cholesterol W1HR BMI FFM Oestradiol Age B LDL-cholesterol W1HR BMI FFM Oestradiol Glucose were found with LDL-cholesterol values, which were significantly and positively correlated with W1HR and BMI, and negatively with FFM, oestradiol, and glucose values. Although HDL-cholesterol levels did not significantly differ amongst the groups, they showed a significant and positive correlation with age, the glucose:insulin molar ratio, SHBG and testosterone and a negative correlation with W1HR and BMI values (multiple r 0.502; P 0.001). Discussion It is commonly accepted that altered lipid profiles may help to explain why postmenopausal women appear to be more susceptible to atherosclerotic cardiovascular events regardless of the effects of age [19]. This study indicates that menopausal status per se may have adverse effect so on lipid profiles in middle-aged women, because postmenopausal women had significantly higher concentrations of total and LDL-cholesterol with respect to pre- and perimenopausal women. Mechanisms responsible for increased cholesterol concentrations in the early postmenopausal period are far from being clear. As reported in previous epidemiological studies [19], we found that both total and LDL-cholesterol levels were significantly correlated with BMI, regardless of the effects of menopause on body weight and body composition. There is also epidemiological evidence that higher than normal cholesterol levels may be associated with abdominal body fat distribution in either obese and nonobese individuals [20]. However, it is still controversial whether menopausal status per se may affect fat distribution [21 24]. We have previously reported that an increase of the WHR ratio, which occurs with increasing age in middle-aged women, seems mainly to depend on BMI and age variations and not on menopause or related normal pattern [8]. Nonetheless, the significant relationship we found between W1HR and total or LDL-cholesterol suggests a significant age-independent effect of the pattern of fat distribution upon cholesterol metabolism. Changes in blood cholesterol were also inversely correlated with the amount of FFM regardless of the effects of body weight and fat mass. We suggest that the inverse correlation between FFM and total cholesterol does not imply a cause effect relationship, but it may depend on the coordinated effects of other factors, including age and androgens. Oestradiol concentrations were another factor significantly and negatively correlated with total and DL cholesterol levels. This supports the concept that oestrogens may play an independent role in the

8 202 RENATO PASQUALI et al. regulation of lipid metabolism in women. It is well known that oestrogens induce favourable changes in the plasma lipoprotein profiles in postmenopausal women. They do in fact increase HDL-cholesterol concentrations, particularly the HDL2 subfraction [25] and the apo A-1 synthetic rates [26]. Moreover they may favour the decrease of LDH-cholesterol levels [6, 7] and up-regulate LDL receptor density [27]. These effects may partially disappear during the menopause, because of the decrease in oestradiol production rates, blood concentrations, and tissue delivery [6]. On the other hand, it has been extensively demonstrated [7, 27] that in postmenopausal women hormonal replacement may have beneficial effects on lipid metabolism, in particular by decreasing LDL-cholesterol and increasing HDL-cholesterol concentrations [6]. Taken together therefore, all these data give further support to the hypothesis that, amongst others, the fall in oestrogen concentration may represent an important factor responsible for the menopause-related increase of serum cholesterol in middle-aged women. Acknowledgements This study was supported by grants from the Ministry of University and Scientific Research and Technology, , and from the Banca Popolare dell Emilia, Bologna, Italy. We would like to thank the town and particularly the Mayor of Virgilio, Mantua (Italy), for giving us the opportunity to carry out the study and the director of the Carlo Poma Hospital of Mantua for all the assistance to us before and during the study. Special thanks are due to Sergio Ravelli for his support and cooperation throughout the entire period of the survey. References 1 Colditz GA, Willett WC, Stampfer MJ, Rosner B, Speizer FE, Hennekens CH. Menopause and the risk of coronary heart disease in women. 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9 CHOLESTEROL LEVELS AND MENOPAUSAL STATUS 203 use: a cross sectional study in Dutch women participating in the DOM-Project. Int J Ob 1990; 14: Rijpkema AHM, van der Sanden AA, Ruijs AHC. Effects of postmenopausal oestrogen-progestogen replacement therapy on serum lipids and lipoproteins: review. Maturitas 1990; 12: Melchior GW, Casthe CK, Vidmar TJ, Apo-A1 metabolism in cynomolgus monkeys: male-to-female differences. Bioch Bioph acta 1990; 1043: Henriksson P, Stamberger M, Erikson M, Rudling M, Diczfalusy U. Oestrogen-induced changes in lipoprotein metabolism: role in prevention of atherosclerosis in the cholesterol-fed rabbit. Eur J Clin Invest 1989; 19: Received 30 September 1996; accepted 27 October Correspondence: Renato Pasquali MD, Endocrinologia, Dipartimento di Medicina Interna & Gastroenterologia, Policlinico S Orsola-Malpighi, Via Massarenti 9, Bologna, Italy (fax: ).