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Effect of tibolone administration on heart rate variability and free fatty acid levels in postmenopausal women

      Abstract

      Objective: To evaluate the effects of tibolone on heart rate variability and plasma free fatty acid levels in postmenopausal women.
      Design: Randomized, placebo-controlled trial.
      Setting: University hospital.
      Patient(s): Thirty postmenopausal women.
      Intervention(s): Tibolone, 2.5 mg/d, or placebo for 4 months.
      Main Outcome Measures: Variability in heart rate and changes in the lipid profile.
      Result(s): Anthropometric data were unchanged throughout the study. Compared with placebo, long-term tibolone administration was associated with a decrease in plasma levels of low-density lipoprotein cholesterol, triglyceride, and free fatty acid and homeostasis model assessment index. Furthermore, tibolone administration was associated with an increase in RR interval, total power, and high frequency and decrease in low frequency and the low frequency/high frequency ratio. Finally, the δ decrease in plasma free fatty acid levels correlated with δ low frequency/high frequency ratio independently of age, δ body mass index, δ homeostasis model assessment index, and low-density lipoprotein cholesterol levels.
      Conclusion(s): Long-term tibolone administration improves the ratio of cardiac sympathetic tone to parasympathetic tone in postmenopausal women.

      Keywords

      Estrogen replacement therapy decreases risk for cardiovascular mortality (
      • Kannel W.B
      • Hjortland M.C
      • MacNamara P
      Menopause and risk of cardiovascular disease. The Framingham study.
      ,
      • Matthews K.A
      • Meilahn E.N
      • Kuller L.H
      • Kelsey S.F
      • Caggiula A.W
      • Wing R.R
      Menopause and risk factor for coronary heart disease.
      ,
      • Lobo R.A
      Estrogen and cardiovascular disease.
      ,
      • Owens J.F
      • Stoney C.M
      • Matthews K.A
      Menopausal status influences ambulatory blood pressure changes during mental stress.
      ) because it decreases low-density lipoprotein (LDL) cholesterol levels (
      The Writing Group for the PEPI Trial
      Effect of estrogen/progestin regiment on heart disease risk factors in postmenopausal women the Postmenopausal Estrogen/progestin Interventions (PEPI) trial.
      ,
      • Walsh B.W
      • Schiff I
      • Rosner B
      • Greenberg L
      • Ravnikar V
      • Sacks F.M
      Effects of postmenopausal estrogen replacement on the concentrations and metabolism of plasma lipoproteins.
      ), increases high-density lipoprotein (HDL) cholesterol levels (
      The Writing Group for the PEPI Trial
      Effect of estrogen/progestin regiment on heart disease risk factors in postmenopausal women the Postmenopausal Estrogen/progestin Interventions (PEPI) trial.
      ,
      • Walsh B.W
      • Schiff I
      • Rosner B
      • Greenberg L
      • Ravnikar V
      • Sacks F.M
      Effects of postmenopausal estrogen replacement on the concentrations and metabolism of plasma lipoproteins.
      ), inhibits vasoconstrictor response to norepinephrine, and increases parasympathetic tone (
      • Rosano G.M
      • Patrizi R
      • Leonardo F
      • Ponikowski P
      • Collins P
      • Sarre P.M
      • et al.
      Effect of estrogen replacement therapy on heart rate variability and heart rate in healthy postmenopausal women.
      ,
      • Virtanen I
      • Polo O
      • Polo-Kantola P
      • Kuusela T
      • Ekholm E
      The effect of estrogen replacement therapy on cardiac autonomic regulation.
      ,
      • Weitz G
      • Elam M
      • Born J
      • Fehm H.L
      • Dodt C
      Postmenopausal estrogen administration suppresses muscle sympathetic nerve activity.
      ).
      Estrogen may also cause proliferation of endometrial tissue (
      • Whitehead M.I
      • King R.J.B
      • McQueen J
      • Campbell S
      Endometrial histology and biochemistry in climacteric women during oestrogen and oestrogen/progestogen therapy.
      ) and increases the risk for endometrial carcinoma (
      • Gambrell Jr, R.D
      Prevention of endometrial cancer with progestogens.
      ). Combined estrogen-progestin treatment prevents these endometrial abnormalities (
      • Voigt L.F
      • Weiss N.S
      • Chu J
      • Daling J.R
      • McKnight B
      • Van Belle G
      Progestogen supplementation of exogenous oestrogens and risk of endometrial cancer.
      ). Nevertheless, some progesterone-derived agents adversely affect the ratio of LDL to HDL cholesterol (
      • Lobo R.A
      Clinical review 27 effects of hormonal replacement on lipids and lipoproteins in postmenopausal women.
      ), suggesting that the combined therapy may diminish the cardiovascular benefits offered by estrogen alone.
      A good alternative to therapy with estrogen alone is tibolone (
      • Ross L.A
      • Alder E.M
      Tibolone and climacteric symptoms.
      ,
      • Farish E
      • Barnes J.F
      • Rolton H.A
      • Spowart K
      • Fletcher C.D
      • Hart D.M
      Effects of tibolone on lipoprotein(a) and HDL subfractions.
      ,
      • Bjanason N.H
      • Bjarnason K
      • Haarbo J
      • Bennink H.J.T.C
      • Christiansen C
      Tibolone influence on markers of cardiovascular disease.
      ), a synthetic steroid [(7α, 17α)-17 hydroxy-7-methyl-19-norpregn-(10)-en-20-yn-3-one] with estrogenic and some progestogenic and androgenic properties (
      • Ross L.A
      • Alder E.M
      Tibolone and climacteric symptoms.
      ,
      • Farish E
      • Barnes J.F
      • Rolton H.A
      • Spowart K
      • Fletcher C.D
      • Hart D.M
      Effects of tibolone on lipoprotein(a) and HDL subfractions.
      ,
      • Bjanason N.H
      • Bjarnason K
      • Haarbo J
      • Bennink H.J.T.C
      • Christiansen C
      Tibolone influence on markers of cardiovascular disease.
      ). In postmenopausal women, administration of tibolone reduces plasma triglyceride levels (
      • Farish E
      • Barnes J.F
      • Fletcher C.D
      • Ekavall K
      • Calder A
      • Hart D.M
      Effects of tibolone on serum lipoprotein and apolipoprotein levels compared with a cyclical estrogen/progestogen regimen.
      ).
      Because of the strong metabolic link between plasma triglyceride and free fatty acid levels and cardiac sympathetic nervous activity (
      • Paolisso G
      • Manzella D
      • Rizzo M.R
      • Ragno E
      • Barbieri M
      • Varricchio G
      • et al.
      Elevated plasma free fatty acid concentrations stimulate the cardiac autonomic nervous system in healthy subjects.
      ,
      • Manzella D
      • Barbieri M
      • Rizzo M.R
      • Ragno E
      • Passariello N
      • Gambardella A
      • et al.
      Role of free fatty acids on cardiac autonomic nervous system in non insulin-dependent diabetic patients effects of metabolic control.
      ), tibolone may influence cardiac sympathetic nervous activity through a change in plasma free fatty acid concentration. We therefore assessed the efficacy of long-term tibolone administration (2.5 mg/d for 4 months) on plasma free fatty acid concentrations and on cardiac autonomic nervous activity, as assessed by heart rate variability (
      Task Force of the European Society of CardiologyNorth American Society of Paging and Electrophysiology
      Heart rate variability. Standard of measurements, physiological interpretation and clinical use.
      ), in 30 postmenopausal women.

      Materials and methods

      We enrolled 30 healthy postmenopausal women (mean time since the last menstrual period, 32 months [range, 12–83 months]) >50 years of age (mean [±]age, 53±2 years). No participant had received hormonal treatment before the study.
      All patients had no evidence of coronary heart diseases, as confirmed by electrocardiography, echocardiography, and treadmill test. During the study, all women were asked to maintain their body weight, smoking habits, and alcohol and caffeine intake. Insulin resistance was assessed by homeostasis model assessment (HOMA) (
      • Mathews D.R
      • Hosker J.P
      • Rudenski A.S
      • Naylor B.A
      • Treacher D.F
      • Turner R.C
      Homeostasis model assessment insulin resistance and βcell function from fasting plasma glucose and insulin concentrations in man.
      ).
      All tests were performed in the morning and after overnight fasting (for at least 12 hours). The potential risks of the study were clearly explained to each woman, and all participants gave informed consent. They study was approved by the ethical committee of our institution.

      Study protocol

      The study was a randomized, placebo trial. At baseline, all participants were studied at 8:00 a.m. in a quiet, comfortable room that was kept at 22°C to 24°C. A venous blood sample was immediately drawn for plasma metabolite measurement. Each participant rested in the supine position for at least 30 minutes before baseline Holter recording was started. Holter monitoring lasted 60 minutes.
      Patients were then randomly assigned to treatment with tibolone, 2.5 mg/d (Livial; Organon, Rome, Italy) (n = 15) or placebo (sodium citrate) (n = 15). Each treatment lasted 4 months. At the end of treatment period, all patients were reevaluated.

      Anthropometric measurements

      Weight and height were measured by using a standard technique. Body mass index was calculated as body weight in kilograms divided by height in square meters. Waist circumference was measured at the midpoint between the lower rib margin and the iliac crest (normally the umbilical level), and hip circumference was measured at the trochanter level. Both circumferences were measured to the nearest 0.5 cm with a plastic tape measure; the ratio between the two measurements provided the waist-to-hip ratio.
      Anthropometric measures are used because changes in body composition may significantly affect insulin resistance and cardiac autonomic nervous activity. We wanted to exclude the possibility that changes in heart rate variability may have resulted solely from changes in body composition or insulin resistance.

      HOMA index

      Insulin resistance was assessed by using the HOMA index (
      • Mathews D.R
      • Hosker J.P
      • Rudenski A.S
      • Naylor B.A
      • Treacher D.F
      • Turner R.C
      Homeostasis model assessment insulin resistance and βcell function from fasting plasma glucose and insulin concentrations in man.
      ). Homeostasis model assessment (HOMA) is a mathematical model that describes the degree of insulin resistance from a patient’s fasting plasma insulin and glucose concentrations (
      • Mathews D.R
      • Hosker J.P
      • Rudenski A.S
      • Naylor B.A
      • Treacher D.F
      • Turner R.C
      Homeostasis model assessment insulin resistance and βcell function from fasting plasma glucose and insulin concentrations in man.
      ). The accuracy and precision of HOMA have been compared with independent estimates of insulin resistance (
      • Mathews D.R
      • Hosker J.P
      • Rudenski A.S
      • Naylor B.A
      • Treacher D.F
      • Turner R.C
      Homeostasis model assessment insulin resistance and βcell function from fasting plasma glucose and insulin concentrations in man.
      ).

      Data acquisition and analysis

      The software used for data acquisition and analysis is described elsewhere (
      • Paolisso G
      • Manzella D
      • Tagliamonte M.R
      • Rizzo M.R
      • Gambardella A
      • Varricchio M
      Effects of different infusion rates on heart rate variability in lean and obese subjects.
      ,
      • Paolisso G
      • Manzella D
      • Barbieri M
      • Rizzo M.R
      • Gambardella A
      • Varricchio M
      Baseline heart rate variability in healthy centenarians differences compared with aged subjects (>75 years old).
      ). In brief, the computer program first calculates the interval tachogram. From section of tachogram that includes 512 interval values, simple statistics (mean and variance) are calculated. The computer program automatically calculates the autoregressive coefficients needed to define the power spectral density estimate and prints out the power and frequency of every spectral component.
      Two major oscillatory components are usually detectable. One component, which varies synchronous with respiration, is described as high frequency (about 0.25 Hz), and the other component, which corresponds to the slow waves of arterial pressure, is described as low frequency (about 0.1 Hz). Each spectral component is presented as normalized units (nu) by dividing the component by the total power minus the direct current component, if present. Only components comprising >5% of total power were considered significant.
      The low frequency/high frequency ratio is considered an index of balance between cardiac sympathetic and parasympathetic tone (
      Task Force of the European Society of CardiologyNorth American Society of Paging and Electrophysiology
      Heart rate variability. Standard of measurements, physiological interpretation and clinical use.
      ,
      • Malliani A
      • Lombardi F
      • Pagani M
      Power spectral analysis of heart rate variability a tool to explore neural regulatory mechanisms.
      ,
      • Kamath M.V
      • Fallen E.L
      Power spectral analysis of heart rate variability a non-invasive signature of cardiac autonomic function.
      ,
      • Pagani M
      • Montano N
      • Porta A
      Relationship between spectral components of cardiovascular variabilities and direct measures of muscle sympathetic nerve activity in humans.
      ).
      Respiratory frequency over 2 minutes was also calculated before the test. Women with a respiratory rate less than 10 breaths/min (<0.15 Hz) were excluded.

      Analytical techniques

      Plasma glucose concentrations were determined by glucose oxidative methods (glucose autoanalyzer, Beckman Coulter, Inc., Fullerton, CA). Plasma LDL and HDL cholesterol and triglyceride levels were determined by routine laboratory methods. Plasma free fatty acid levels were determined according to the method of Dole et al. (
      • Hanggi W
      • Lippuner K
      • Riesen W
      • Jaeger P
      • Birkhauser M.H
      Long term influence of different postmenopausal hormone replacement regimens on serum lipids and lipoprotein (a) a randomised study.
      ). To avoid in vitro lipolysis, plasma free fatty acid levels were determined in chilled plasma containing ethylenediamine tetraacetic acid and 0.275 mg/mL of paraoxon, a lipoprotein lipase inhibitor (
      • Hanggi W
      • Lippuner K
      • Riesen W
      • Jaeger P
      • Birkhauser M.H
      Long term influence of different postmenopausal hormone replacement regimens on serum lipids and lipoprotein (a) a randomised study.
      ). Plasma insulin levels were determined by radioimmunoassay.

      Statistical analyses

      All results are expressed as the mean (±SD). Mean arterial blood pressure was calculated as diastolic blood pressure plus one third of the pulse pressure. Changes in plasma free fatty acid and triglyceride concentrations, HOMA index, and low frequency/high frequency ratio are used only to compare the changes (not the absolute values) of these variables. Because of the skewed distribution, total power, low frequency, high frequency were logarithmically transformed for statistical testing and back transformed for presentation in table and in figures.
      Analysis of variance was used to analyze differences between the two study groups. Pearson’s simple correlation was used to analyze the association between variables. Partial correlations were used to examine the relation between two variables independently of covariates. P<.05 was considered statistically significant. All calculations were made on an IBM PC computer by using SPSS software, version 10.0 (SPSS, Inc., Chicago, IL).

      Results

      Table 1 shows clinical and laboratory characteristics of the study patients. At baseline, anthropometric, metabolic, and cardiovascular variables were similar in the placebo and tibolone groups. At the end of study, anthropometric, metabolic and cardiovascular variables remained unchanged in the placebo group. In contrast, patients who received tibolone had significant decreases in plasma levels of LDL cholesterol, triglyceride, and free fatty acid in the HOMA index but no change in plasma HDL cholesterol levels (Table 1).
      TABLE 1Anthropometric and metabolic indices after placebo and tibolone administration.
      CharacteristicPlacebo groupTibolone group
      BaselineP valueAfter placeboBaselineP valueAfter tibolone
      Body mass index (kg/m2)26.4 ± 1.3NS26.1 ± 1.826.2 ± 1.2NS26.0 ± 1.6
      Waist-to-hip ratio0.84 ± 0.01NS0.82 ± 0.020.83 ± 0.02NS0.82 ± 0.01
      Glucose level (mmol/L)4.7 ± 0.5NS4.6 ± 0.64.6 ± 0.3NS4.6 ± 0.1
      HDL cholesterol level (mmol/L)2.4 ± 0.1NS2.3 ± 0.32.4 ± 0.3NS2.5 ± 0.1
      LDL cholesterol level (mmol/L)4.8 ± 0.1NS4.7 ± 0.44.8 ± 0.3.033.6 ± 0.1
      P<.05 for placebo vs. tibolone.
      Triglyceride level (mmol/L)0.76 ± 0.06NS0.78 ± 0.020.75 ± 0.10.010.58 ± 0.09
      P<.03 for placebo vs. tibolone.
      Free fatty acid level (mmol/L)554 ± 38NS557 ± 43555 ± 36.01388 ± 33
      P<.03 for placebo vs. tibolone.
      HOMA index1.91 ± 0.11NS1.90 ± 0.101.89 ± 0.12.051.52 ± 0.14
      P<.01 placebo vs. tibolone.
      Mean arterial blood pressure (mm Hg)101 ± 5NS101 ± 3100 ± 6NS101 ± 5
      Note: Results are the mean (±SD). All metabolites were stained during fasting. HDL = high-density lipoprotein; HOMA = homeostasis model assessment; LDL = low-density lipoprotein.
      Manzella. Effect of tibolone on heart rate. Fertil Steril 2002.
      a P<.05 for placebo vs. tibolone.
      b P<.03 for placebo vs. tibolone.
      c P<.01 placebo vs. tibolone.
      Tibolone administration was associated with a significant increase in RR interval, total power, and high frequency and a decrease in low frequency and the low frequency/high frequency ratio (Table 2). Because differences between plasma free fatty acid levels and low frequency/high frequency ratio differed significantly after treatment with tibolone, delta (δ) changes in those variables were calculated to compare them more appropriately. In the tibolone group, δ changes in plasma free fatty acid levels correlated with δ changes in the low frequency/high frequency index (r=0.72; p<0.01). This correlation persisted even after adjustment for age, δ change in body mass index, δ change in HOMA index, and δ change in LDL cholesterol levels (Fig. 1).
      TABLE 2Heart rate variability parameters after placebo and tibolone administration.
      VariablePlacebo groupTibolone group
      BaselineP valueAfter placeboBaselineP valueAfter tibolone
      RR interval (msec)760 ± 12NS761 ± 14761 ± 150.05846 ± 22
      P<.05 for placebo vs. tibolone.
      Total power (msec2)2739 ± 442NS2712 ± 4212715 ± 4520.052991 ± 494
      P<.05 for placebo vs. tibolone.
      Low frequency (nu)68.5 ± 2.1NS67.4 ± 1.367.3 ± 1.50.0350.1 ± 1.8
      P<.03 for placebo vs. tibolone.
      High frequency (nu)20.1 ± 2.6NS20.3 ± 1.119.8 ± 2.40.0539.4 ± 1.6
      P<.03 for placebo vs. tibolone.
      Low frequency/high frequency ratio5.7 ± 0.2NS5.5 ± 0.65.8 ± 0.40.011.5 ± 0.3
      P<.01 for placebo vs. tibolone.
      Note: All results are the mean (±SD).
      Manzella. Effect of tibolone on heart rate. Fertil Steril 2002.
      a P<.05 for placebo vs. tibolone.
      b P<.03 for placebo vs. tibolone.
      c P<.01 for placebo vs. tibolone.
      Figure thumbnail GR1
      FIGURE 1Partial correlation between δ decrease in plasma free fatty acid (FFA) concentration and δ decrease in low frequency/high frequency (LF/HF) ratio in women who received tibolone (n = 15).
      Manzella. Effect of tibolone on heart rate. Fertil Steril 2002.

      Discussion

      Our study demonstrates that long-term administration of tibolone decreases plasma levels of free fatty acid and has a positive effect on cardiac autonomic nervous activity in postmenopausal women.
      Estrogen replacement therapy may decrease cardiovascular mortality in postmenopausal women in part by decreasing plasma levels of total cholesterol and LDL cholesterol and increasing plasma HDL cholesterol levels (
      The Writing Group for the PEPI Trial
      Effect of estrogen/progestin regiment on heart disease risk factors in postmenopausal women the Postmenopausal Estrogen/progestin Interventions (PEPI) trial.
      ,
      • Walsh B.W
      • Schiff I
      • Rosner B
      • Greenberg L
      • Ravnikar V
      • Sacks F.M
      Effects of postmenopausal estrogen replacement on the concentrations and metabolism of plasma lipoproteins.
      ). However, estrogen replacement therapy is also associated with an increase in plasma triglyceride levels (
      • Hanggi W
      • Lippuner K
      • Riesen W
      • Jaeger P
      • Birkhauser M.H
      Long term influence of different postmenopausal hormone replacement regimens on serum lipids and lipoprotein (a) a randomised study.
      ). Concomitant administration of a progestin can blunt the improvement in serum lipid levels provided by estrogen (
      • Lobo R.A
      Clinical review 27 effects of hormonal replacement on lipids and lipoproteins in postmenopausal women.
      ).
      In contrast, tibolone decreases plasma triglyceride levels (
      • Farish E
      • Barnes J.F
      • Fletcher C.D
      • Ekavall K
      • Calder A
      • Hart D.M
      Effects of tibolone on serum lipoprotein and apolipoprotein levels compared with a cyclical estrogen/progestogen regimen.
      ). This effect may be useful to prevent cardiovascular mortality in postmenopausal women. The decrease in plasma levels of triglyceride and free fatty acid provides a further pathophysiologic mechanism that may explain the relationship between menopause and cardiac mortality, since plasma free fatty acid levels negatively affect the sympathetic nervous system.
      The relationship between plasma free fatty acid levels and the autonomic nervous system was suggested by Bulow et al. (
      • Bulow J
      • Modsen J
      • Astrup P
      • Christiansen N.J
      Vasoconstrictor effect of high free fatty acids/albumin ratio in adipose tissue in vivo.
      ), who used lipid emulsion plus heparin to increase blood pressure and total peripheral resistance in pigs. Although the mechanism of this effect was not elucidated, these investigators had previously shown that local perfusion of adipose tissue with free fatty acid causes vasoconstriction (
      • Bulow J
      • Modsen J
      • Astrup P
      • Christiansen N.J
      Vasoconstrictor effect of high free fatty acids/albumin ratio in adipose tissue in vivo.
      ). Later, Stepniakowski et al. (
      • Stepniakowski K
      • Goodfriend T.L
      • Egan B.M
      Fatty acids enhance vascular alpha-adrenergic sensitivity.
      ) reported that infusion of lipid emulsion plus heparin reduced vein distensibility in healthy volunteers and increased responsiveness to phenylephrine. The latter observation suggested a direct pressor effect of fatty acids on vascular beds.
      Grekin et al. (
      • Grekin R.J
      • Vollmer A.P
      • Sider R.S
      Pressor effect of portal venous oleate infusion. A proposed mechanism for obesity hypertension.
      ) reported that portal free fatty acid infusion also has significant pressor effects that may be mediated by increased sympathetic tone. It has also been demonstrated that elevated plasma free fatty acid levels may stimulate cardiac sympathetic nervous system in healthy persons (
      • Paolisso G
      • Manzella D
      • Rizzo M.R
      • Ragno E
      • Barbieri M
      • Varricchio G
      • et al.
      Elevated plasma free fatty acid concentrations stimulate the cardiac autonomic nervous system in healthy subjects.
      ) by increasing plasma catecholamine levels. The latter effect has also been confirmed in patients with type 2 diabetes mellitus (
      • Manzella D
      • Barbieri M
      • Rizzo M.R
      • Ragno E
      • Passariello N
      • Gambardella A
      • et al.
      Role of free fatty acids on cardiac autonomic nervous system in non insulin-dependent diabetic patients effects of metabolic control.
      ).
      To our knowledge, our study is the first to show that tibolone administration is associated with a decrease in plasma free fatty acid levels and beneficial modulation of cardiac autonomic activity in terms of the low frequency/high frequency ratio. We also show that positive modulation of cardiac autonomic nervous system is not influenced by degree of insulin resistance or distribution of body fat. The association between plasma free fatty acid levels and low frequency/high frequency ratio remained after adjustment for such confounding factors as age, body mass index, waist-to-hip ratio, and HOMA index.
      A possible limitation of our study is the short duration of treatment and small sample. Larger studies of longer-term administration of tibolone are needed to confirm and extend our findings.
      In conclusion, prolonged administration of tibolone was associated with a significant decrease in plasma triglyceride and free fatty acid levels and with rebalance of cardiac autonomic nervous activity in postmenopausal women. Our results are especially relevant in light of the data showing that postmenopausal women experience a significant increase in cardiac sympathetic activity (
      • Huikuri H.V
      • Pikkujamsa S.M
      • Airaksinen K.E.J
      • Ikanheimo M.J
      • Rantala A.O
      • Kauma H
      • et al.
      Sex-related differences in autonomic modulation of heart rate in middle-aged subjects.
      ,
      • Rosano G.M.C
      • Patrizi R
      • Leonardo F
      • Ponikowshki P
      • Collins P
      • Sarre P.M
      • et al.
      Effect of estrogen replacement therapy on heart rate variability and heart rate in healthy postmenopausal women.
      ,
      • Du X.J
      • Riemersma R.A
      • Dart A.M
      Cardiovascular protection by estrogens is partly mediated through modulation of autonomic nervous function.
      ) and that myocardial infarction and heart failure are normally associated with sympathetic overactivity (
      • Bigger J.T
      • Fleiss J.L
      • Steinman R.C
      • Rolnitzky L.M
      • Kleiger R.E
      • Rottman J.N
      Frequency domain measures of heart period variability and mortality after myocardial infarction.
      ,
      • Barron H.V
      • Lesh M.D
      Autonomic nervous system and sudden cardiac death.
      ). Epidemiologic studies should consider plasma free fatty acid levels as an important variable that affect the cardiac mortality risk in postmenopausal women.(
      • Dole V.P
      • Meinertz H
      Micro-determination of long-chain fatty acids in plasma and tissues.
      )

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