Relations of plasma homocysteine to left ventricular structure and function: the Framingham Heart Study

Johan Sundströma, Lisa Sullivand, Jacob Selhube, Emelia J Benjamina,b,c, Ralph B D'Agostinod, Paul F Jacquese, Irwin H Rosenberge, Daniel Levya,b,f, Peter W.F Wilsona and Ramachandran S Vasana,b,c,*

a The Framingham Heart Study, Framingham, MA, USA
b Department of Preventive Medicine, Boston University School of Medicine, Boston, MA, USA
c Cardiology Section, Boston University School of Medicine, Boston, MA, USA
d Department of Mathematics, Boston University, Boston, MA, USA
e The Jean Mayer–USDA Human Nutrition Research Center on Aging, Tufts University, Boston, MA, USA
f The National Heart, Lung, and Blood Institute, USA

Received August 29, 2003; revised January 3, 2004; accepted January 15, 2004 * Corresponding author. Tel.: +1-5089353450; fax: +1-5086261262
E-mail address: vasan{at}fram.nhlbi.nih.gov


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Conclusions
 Appendix A
 Echocardiography quality control
 References
 
Aims Hyperhomocysteinaemia is a risk factor for congestive heart failure, especially in women. We investigated if homocysteine promotes left ventricular (LV) remodelling.

Methods and results We examined cross-sectional relations of plasma total homocysteine to echocardiographic LV structure and function in 2697 Framingham Heart Study participants (mean age 58 years, 58% women) free of heart failure and previous myocardial infarction. Adjusting for age and height, plasma homocysteine was positively related to LV mass, wall thickness, and relative wall thickness in women (–0.04), but not in men (–0.68). Adjusting additionally for other clinical covariates, the relations of plasma homocysteine to LV mass and wall thickness in women remained statistically significant, but the relation to relative wall thickness became of borderline significance (1.92 g, 0.01 cm, and 0.29% increase, respectively, for a 1-SD increase in ln[homocysteine], –0.08). LV mass and wall thickness were higher in the fourth quartile of plasma homocysteine compared to the lower three in all models in women (–0.02), but not in men (–0.78). Plasma homocysteine was not related to left atrial size or LV fractional shortening in either sex.

Conclusion In our community-based sample, plasma homocysteine was directly related to LV mass and wall thickness in women but not in men.

Key Words: Heart failure • Left ventricular hypertrophy • Left ventricular remodelling • Metabolism • Echocardiography


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Conclusions
 Appendix A
 Echocardiography quality control
 References
 
Plasma homocysteine is a modest risk factor for cardiovascular morbidity and mortality.1 More recently, plasma homocysteine has been shown to be a risk factor for the development of congestive heart failure in individuals free of myocardial infarction, especially in women.2 Homocysteine can also cause cardiac dysfunction through ischaemic mechanisms.1 The pathophysiological mechanisms by which hyperhomocysteinaemia promotes congestive heart failure are not completely known. Hyperhomocysteinaemia can induce left ventricular (LV) hypertrophy and cardiac fibrosis in animal models that is reversed with folate treatment.3,4 In humans, plasma homocysteine has been reported to be increased in hypertensive persons5–7 and has been related to LV hypertrophy in subjects with end-stage renal disease8 but not in a limited sample of hypertensive subjects.9

No prior investigation has evaluated the relations of plasma homocysteine to echocardiographic measures of LV structure and function in the general population. Such an investigation is crucial to examine the hypothesis that hyperhomocysteinaemia may be a risk factor for heart failure via promotion of LV remodelling. Based on evidence described above, we hypothesised that plasma homocysteine is associated with increased LV mass and wall thickness and that these relations may differ in women and men. Accordingly, we examined sex-specific cross-sectional relations of plasma homocysteine to echocardiographic indices of LV structure and function in Framingham Heart Study participants.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Conclusions
 Appendix A
 Echocardiography quality control
 References
 
Study sample
The design and selection criteria of the Framingham Offspring Study have been described previously.10 Participants in this longitudinal study are predominantly Caucasian and are examined every four years. The 3722 participants at the sixth examination cycle (1995–1998) were eligible for the present study.

Participants were excluded if they had a history of congestive heart failure () or recognised myocardial infarction (), serum creatinine 2.0 mg/dl (), or missing echocardiographic measurements (). After these exclusions, 2697 participants remained eligible (57.5% women). The persons included were younger, more likely to be female, and generally in better health (Table 3). The study complies with the Declaration of Helsinki and was approved by the Institutional Review Board at Boston Medical Center. All subjects gave written informed consent.


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Table 3 Characteristics of participants with complete and missing echocardiographic data

 
Clinical and laboratory examinations
Participants underwent a standardised medical history and physical examination, including measurements of blood pressure and phlebotomy after an overnight fast for laboratory tests. Total plasma homocysteine was measured at the sixth examination cycle using high performance liquid chromatography with fluorometric detection.11 The coefficient of variation for this assay was 9%.12 Diabetes and hypertension were defined according to current guidelines.13,14 Prevalence or history of congestive heart failure or recognised myocardial infarction were established by a panel of three physicians.15

Echocardiographic methods
Participants underwent routine transthoracic echocardiography with Doppler colour flow imaging. M-mode measurements of LV dimensions were obtained using the leading edge-to-leading edge convention.16 Interventricular septum thickness (IVS), posterior LV wall thickness (PW), and LV end-diastolic diameter (LVEDD) were measured at end-diastole and LV end-systolic diameter also at end-systole (LVESD). LV wall thickness (LVWT) was calculated as IVS+PW, and LV relative wall thickness was calculated as (IVS+PW)/LVEDD. LV mass was calculated as 0.8[1.04(IVS+LVEDD+PW)3–(LVEDD)3]+0.6.17 LV fractional shortening was calculated as (LVEDD-LVESD)/LVEDD. Valve disease was defined as more than a mild degree of aortic or mitral stenosis or regurgitation on colour Doppler echocardiography. In secondary analyses, the LV ejection fraction determined by visual estimation on multiple views by an experienced echocardiographer was used (normal LV systolic function: ejection fraction >=50%, LV systolic dysfunction: ejection fraction 50%).18 Inter- and intra-reader correlations of echocardiographic measurements were excellent (see Table 4).


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Table 4 Reproducibility of echocardiographic measurements at sixth Offspring examination

 
Statistical analyses
We initially investigated distributional properties of all study variables for the total sample and separately for men and women. Because the distribution of plasma homocysteine values was non-normal, we logarithmically transformed the values to promote normality for statistical modelling. Multiple linear regression models were used to examine relations of echocardiographic measures to plasma homocysteine. Plasma homocysteine was assessed as a continuous variable (natural logarithm), and as a categorical variable (sex-specific quartiles). In the latter models, we tested for a linear trend for increase in LV measures across quartiles of plasma homocysteine, and for any evidence of a threshold where the top quartile was associated with increased LV measures relative to the lower three. Three sets of multivariable models were examined in a hierarchical fashion:
  1. Adjusted for age and height.
  2. Adjusted for age, height, systolic blood pressure, and antihypertensive treatment.
  3. Adjusted for age, height, weight, total/HDL-cholesterol ratio, systolic blood pressure, antihypertensive treatment, diabetes, valve disease, heart rate, and serum creatinine.
In each of these models, for each echocardiographic outcome, we examined the regression coefficients associated with plasma homocysteine as measures of effect and also summarised the means of the echocardiographic measurements across quartiles of plasma homocysteine.

We performed a series of analyses of residuals from regression models. We assessed the distributions of residuals in univariate plots (e.g., histograms to assess normality) and then in two-dimensional plots of residuals by predicted values to assess the appropriateness of the regression assumptions. In each of our models these plots suggested that the assumptions of linearity and homogeneity of variance were met.

All analyses were sex-specific and were defined a priori. A formal interaction term between plasma homocysteine and sex was tested in multivariable models A and found to be statistically significant for LV mass (), wall thickness (), and relative wall thickness (), suggesting effect modification by gender and further emphasizing the importance of sex-specific analyses.

In secondary analyses, we examined if the relations of plasma homocysteine to echocardiographic measurements varied according to age, systolic blood pressure, body mass index, and menopausal status by incorporating interaction terms in model B and by stratifying by menopausal status. A covariate for examination date (before/after mandatory folate fortification of cereal was introduced) was also investigated in all models. We also repeated all analyses in a subset of participants who did not have any LV systolic dysfunction (as defined above) at baseline. These analyses paralleled the principal analyses (using log-transformed plasma homocysteine and quartiles, incorporating covariates noted above).

Multiple logistic regression models were used to investigate relations of plasma homocysteine to LV systolic dysfunction (dichotomous LV ejection fraction). Odds ratios and their 95% confidence limits were estimated for the presence of LV systolic dysfunction for each SD increment in log-transformed plasma homocysteine.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Conclusions
 Appendix A
 Echocardiography quality control
 References
 
Primary analyses
The mean±SD plasma homocysteine was 9.6±3.9 µmol/l in the study sample. Clinical characteristics of the study participants are shown in Table 1.


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Table 1 Characteristics of study sample

 
In multiple linear regression models adjusting for age and height (Table 2, model A), plasma homocysteine was positively related to LV mass, wall thickness, and relative wall thickness (2.91 g, 0.02 cm, and 0.34% increase, respectively, relative to a 1-SD increase in ln[homocysteine], –0.04) in women, but not in men (–0.68). When systolic blood pressure and anti-hypertensive treatment were incorporated into the models (Table 2, model B), there was some attenuation of the associations noted above; statistical significance was still robust for LV mass and wall thickness, but the relations to relative wall thickness became borderline significant (2.27 g, 0.016 cm, and 0.29% increase, respectively, relative to a 1-SD increase in ln[homocysteine], , 0.003 and 0.07, respectively) in women. In multivariable models adjusting for all clinical covariates (Table 2, model C), in women the relations of plasma homocysteine to LV mass and wall thickness remained statistically significant, but the relation to relative wall thickness was of borderline statistical significance (1.92 g, 0.01 cm, and 0.29% increase, respectively, for a 1-SD increase in ln[homocysteine], –0.08).


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Table 2 Relations of plasma homocysteine to echocardiographic left ventricular measures

 
Least-squares means of adjusted echocardiographic LV measures across quartiles of plasma homocysteine are shown in Table 2. In models adjusted for age and height (model A), LV mass and LV wall thickness increased across quartiles of plasma homocysteine in women (–0.01), but not in men (–0.42). After adjustment for clinical covariates (models B and C), the relations of LV mass and wall thickness to plasma homocysteine quartiles in women were slightly attenuated (–0.05).

In threshold models comparing echocardiographic measures in the fourth quartile of plasma homocysteine with those of the lower three quartiles, we observed significantly higher LV mass and wall thickness in the top quartile in age- and height-adjusted (model A) and multivariable-adjusted models (models B and C) in women (–0.02), but not in men (–0.78).

Plasma homocysteine was not related to left atrial size (data not shown), LV fractional shortening, or LVEDD in either sex in any of the models.

Secondary analyses
None of the interaction terms investigated (homocysteinex[age; systolic blood pressure; body mass index]) was found to be statistically significant. The variable for examination date was not statistically significant in any model. The relations of plasma homocysteine to echocardiographic LV measures in women did not vary with menopausal status.

Seventy-six subjects (12 women) had LV systolic dysfunction without overt congestive heart failure. Plasma homocysteine was related to prevalent asymptomatic LV systolic dysfunction in men in age- and height-adjusted models (odds ratio 2.39 [95% confidence interval 1.03–5.53] relative to a 1-SD increase in ln[homocysteine]), but not in multivariable-adjusted models (odds ratio 2.13 [0.37–12.17]). The number of women with asymptomatic LV systolic dysfunction was too small to analyse.

When analyses were repeated excluding the 76 subjects with asymptomatic LV systolic dysfunction, results in men remained unaltered (no association of plasma homocysteine with LV measures was observed). However, in women the association of plasma homocysteine with LV mass, wall thickness, and relative wall thickness strengthened in some models (data not shown).

Statistical power
Because of the smaller number of men in our sample, we investigated our statistical power to detect associations of plasma homocysteine with LV measures in men. The size effect considered in these calculations was based on the difference in LV mass that we observed in women: a difference of 6 g comparing the highest quartile to the lower three quartiles, a standard deviation of LV mass of 32 g, with a size effect of 0.19. Using analysis of variance and our sample size of 1145 men, we had 85% power to detect differences in LV mass comparable to the differences observed in women between the highest and the lower three quartiles of plasma homocysteine in men.


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Conclusions
 Appendix A
 Echocardiography quality control
 References
 
The present study reports the cross-sectional relations of plasma homocysteine to echocardiographic LV measures for the first time in a large community-based sample. Our principal finding is that log-transformed plasma homocysteine was independently associated with LV mass and LV wall thickness in women but not in men. In models evaluating plasma homocysteine quartiles, we observed an apparent threshold effect at the fourth quartile. It is noteworthy that the cutoff point for quartile four was 10.3 µmol/l in women, which is noticeably lower than the commonly used upper limit of normality of 14 µmol/l. Plasma homocysteine was not related to left atrial size, LV fractional shortening, or LVEDD in either sex.

Mechanisms
Hyperhomocysteinaemia may promote LV hypertrophy through vascular and non-vascular mechanisms. Homocysteine has growth-promoting and collagen production-stimulating effects on vascular smooth muscle cells and inhibitory effects on endothelial cell growth.19–21 Homocysteine induces oxidative stress and activates matrix metalloproteinases22 causing endothelial and structural vascular dysfunction,23,24 which ultimately leads to atherosclerosis. In addition to the vascular effects, homocysteine has been demonstrated to have direct adverse effects on the myocardium in experimental settings, affecting the extracellular matrix more than the cardiomyocyte compartment.3,4 Homocysteine directly promotes cardiac fibrosis and cardiac matrix metalloproteinase activity,3,4 resulting in isolated diastolic dysfunction.3 In humans, plasma homocysteine has previously been cross-sectionally associated with LV mass in the setting of end-stage renal disease.8

Gender differences
The gender difference observed in the present study has not been reported previously, although previous studies were conducted in small, selected referral samples and thus had limited power to detect such a difference.8,9 In the present study, we were adequately powered to detect modest associations of homocysteine to LV hypertrophy in both men and women.

In a recent investigation, plasma homocysteine was related in a continuous fashion to heart failure incidence in women, but the association was noted only above the median value of homocysteine in men.2 The findings of the present study support the existence of a gender-related difference in the relations between homocysteine and cardiac remodelling, and subsequent heart failure. The gender difference observed in the relation of plasma homocysteine to LV hypertrophy may contribute to the greater propensity of women to develop heart failure with mainly diastolic dysfunction but normal LV systolic function.

Gender differences in relations of plasma homocysteine to other clinical outcomes have been reported previously, with a higher risk for cardiovascular disease associated with hyperhomocysteinaemia in women compared with men.25 Because LV mass is a risk factor for cardiovascular disease, our data may also provide an explanation for these observations.

In the Third National Health and Nutrition Examination Survey,6 plasma homocysteine was associated with increased prevalence of hypertension in women, but not men. In the present study, plasma homocysteine was associated with increased LV mass in women even after accounting for the influence of blood pressure. Observations of gender differences in relations of homocysteine to LV measures, but not to carotid artery atherosclerosis,26 suggest that non-vascular LV remodelling mechanisms may be important and may vary by gender.

Sex differences in responses to pressure overload have been reported previously with a greater degree of increase in LV wall thickness and concentric hypertrophy noted in women than in men.27 It has been postulated that molecular differences in the LV remodelling process in the two sexes may underlie these observations.28 It is intriguing that homocysteine, like digoxin, is an inhibitor of Na+–K+-ATPase.29 Digoxin treatment has been reported to be associated with more adverse outcomes in women than in men with heart failure.30 It is conceivable that inhibition of Na+–K+-ATPase by homocysteine may promote adverse LV remodelling to a greater extent in women compared to men.2

Oestrogen has been proposed to favourably influence LV remodelling,31 and may also lower plasma homocysteine levels.32 In this context, postmenopausal women with lower oestrogen levels may be more prone to adverse effects of homocysteine on LV remodelling. However, in our sample, the relations of plasma homocysteine to LV measures did not vary with menopausal status.

Strengths and limitations
The strengths of our investigation include a large community-based sample, the standardised measurement of plasma homocysteine, and the statistical power to perform sex-specific analyses. An important limitation is that LV diastolic function was not assessed. Further, M-mode measurements of LV dimensions may be prone to error when the LV configuration is distorted. We minimised this problem by excluding subjects with myocardial infarction and heart failure. Our sample was predominantly Caucasian and the generalisability to other ethnic groups is unknown. Confirmation of this report from other similar investigations will be important. The introduction of folate fortification of cereal during the study period lowered high plasma homocysteine concentrations33 and may have weakened the observed associations.


    Conclusions
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Conclusions
 Appendix A
 Echocardiography quality control
 References
 
Plasma homocysteine was directly related to LV mass and wall thickness in women but not in men in our community-based sample. These observations may underlie the stronger relations of plasma homocysteine to congestive heart failure and cardiovascular disease in women relative to men noted in previous reports.2,25 Additional investigations are warranted to confirm these findings and to elucidate the biological underpinnings of these sex-related differences.


    Appendix A
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Conclusions
 Appendix A
 Echocardiography quality control
 References
 
See Table 3.


    Echocardiography quality control
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Conclusions
 Appendix A
 Echocardiography quality control
 References
 
Echocardiograms at the sixth Offspring examination were read by one of two sonographers or a cardiologist with expertise in echocardiography. Routine quality control measures in the Framingham echocardiography laboratory include strategies to optimise image quality and acquisition, limiting the number of readers, standardised training of readers, periodic blind duplicate readings of a calibration set, reader review sessions, and assessment of temporal reproducibility of measurements. At examination six, twenty echocardiography studies were chosen as a `calibration set' for assessment of intra- and inter-reader reliability. Each study was read by four readers (physicians and echo technicians) with replicate readings done about one month later. Excellent inter-reader and intra-reader correlations of echocardiographic measurements were observed at the index examination, and the mean values of various measurements were quite consistent across the 4 years of the examination cycle (see Table 4).


    Acknowledgments
 
NHLBI/NIH Contracts #N01-HC-25195, 1R01HL67288-01, 5R01-NS-17950, R01HL71039, N01-HV-28178 and 1K24HL04334 (Dr. Vasan), and the Swedish Heart Lung Foundation (Dr. Sundström). This project has been funded in part with Federal funds provided by the US Department of Agriculture under agreement No. 58-1950-9-001. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the authors and do not necessarily reflect the views or policies of the US Department of Agriculture, nor does mention of trade names, commercial products, or organisations imply endorsement by the US Government. No conflicts of interest exist.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Conclusions
 Appendix A
 Echocardiography quality control
 References
 

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