Low serum lycopene and β-carotene increase risk of acute myocardial infarction in men

Abstract

Objective: Previous studies have shown that high intake or concentrations of serum carotenoids may protect against acute myocardial infarction (AMI). The role of carotenoids on the risk of AMI remains inconsistent. The aim of the present study was to examine if serum concentrations of major carotenoids are related to AMI in men. Methods: The study population consisted of 1031 Finnish men aged 46–65 years in the Kuopio Ischaemic Heart Disease Risk Factor (KIHD) cohort. Serum concentrations of carotenoids, retinol and α-tocopherol were measured by high-performance liquid chromatography. The association between the serum concentrations of lycopene α-carotene and β-carotene and the risk of AMI was studied by using the Cox proportional hazard models. Results: A total of 194 incident AMI cases occurred during an average of 11.5 follow-up years. After adjusting for potential confounders, the risk of AMI for men in the lowest tertile of serum concentrations compared with men in the highest tertile was 1.55 (95% CI 1.05– 2.30; P = 0.028) for lycopene and 1.60 (95% CI 1.09–2.35; P = 0.017) for β-carotene. Conclusions: This cross-sectional study shows that low serum lycopene and β-carotene concentrations may increase the risk of AMI in men.

Introduction

Smoking cessation, control of body weight and hypertension, treatment of diabetes mellitus and hyperlipidemia are the main options for primary prevention of coronary heart disease (CHD).¹ Common antioxidants including carotenoids could be used for prevention of atherosclerotic cardiovascular diseases and CHD.² Studies have shown that fruits and vegetables contain carotenoids, which scavenge free radicals under oxidative stress and thereby prevent oxidative modification of low-density lipoprotein (LDL), development and progression of atherosclerosis. Progression of atherosclerosis can lead to CHD and increase risk of myocardial infarction (MI).^3,4

Several previous epidemiological studies have shown that high dietary intake or plasma levels of carotenoids are inversely related to cardiovascular events.⁵ It has been reported that high plasma or adipose tissue concentrations or dietary intake of carotenoids may protect against the risk of acute myocardial infarction (AMI).^6–13 However, some studies have not found association between dietary intake and plasma levels of carotenoids and (AMI).^5,14,15 The aim of this study was to find out whether serum carotenoids may protect against AMI.

Methods

Study population

The KIHD is an ongoing, population-based, prospective cohort study primarily designed to investigate risk factors for cardiovascular diseases (CVD) and related outcomes in middle-aged men from Eastern Finland. The study protocol was approved by the Research Ethics Committee of the University of Kuopio. All study subjects gave their written informed consent. A total of 2682 participants living in Kuopio and its rural communities (82.9% of those eligible), aged 42, 48, 54 or 60 years, were enrolled in the baseline examinations between March 1984 and December 1989.¹⁶ Four-year re-examinations (the entry of this study) for those examined in 1986–1989 were conducted between March 1991 and December 1993. For the reexaminations, the subjects visited the study site twice, with an interval of 1 week. Blood pressure was measured at the first visit and blood samples were drawn at the second visit. Of the 1177 eligible men, 139 were excluded because of death, serious disease, a previous history of (AMI) or missing AMI data. Of the remaining 1038 participants (88.3%), data on serum lycopene and β-carotene concentrations were available for 1031 men.

Biochemical analyses

Blood specimens were collected in Terumo Venoject vacuum tubes (Terumo, Tokyo) from the antecubital vein without tourniquet after an overnight fast. Subjects had rested in a supine position for 30 min before blood sampling. Subjects were instructed to abstain from consuming alcohol for 3 days and from smoking for 12 h before blood collection. Serum for carotenoids and other biochemical measurements were frozen immediately after separation. All blood specimens were collected at baseline before the diagnoses of AMI.

Lycopene, retinol, α-tocopherol, α-carotene and β-carotene serum concentrations have been measured from frozen serum that had been stored at −80°C for 4–36 months by using a modification of the HPLC method of Thurnham et al.¹⁷ Briefly, 200 µl of serum was extracted with 5 ml of hexane and 1 ml of ethanol. The hexane layer was separated and evaporated to dryness under nitrogen at room temperature and the residue was dissolved in 200 µl of the mobile phase (acetonitrile–methanol–chloroform 47:47:6, v/v/v). Samples were injected in a C₁₈ analytical column at room temperature. Peaks were detected at wavelengths of 470 nm for lycopene, at 454 nm for other carotenoids, at 325 nm for retinol and at 292 nm for α-tocopherol by a diode array detector (Model 168; Beckman Instruments, San Ramon, CA, USA). Pure analytes from Sigma (St Louis, MO, USA) were used as primary standards and their concentrations were determined by spectrophotometer. As the stability of the pure carotenoids is poor, a frozen serum pool was used as the secondary standard with the analysis batches. The detection limits and inter-assay CVs have been described earlier.¹⁸ The values below the detection limits of the assay were marked as 0.00 for the statistical analysis.

Concentrations of serum total and LDL cholesterol and triglycerides were analysed with enzymatic methods (Thermo Fisher Scientific, Vantaa, Finland). Serum HDL cholesterol was measured after magnesium chloride dextran sulphate precipitation from the supernatant with enzymatic method (Thermo Fisher Scientific).

Covariates

Resting blood pressure was measured in the morning by two trained nurses with a random-zero mercury sphygmomanometer (Hawksley, Lancing, UK). After the subjects had rested in a supine position for 5 min, 6 measurements were taken at 5 min intervals: 3 while the subjects were in a supine position, 1 while the subjects were standing and 2 while the subjects were sitting. The mean of all 6 measurements was used as the systolic blood pressure and diastolic blood pressure. Body mass index (BMI) was computed as the ratio of weight (kilograms) to the square of height (meters). Alcohol consumption was assessed with a structured quantity–frequency method on drinking behaviour over the previous 12 months. Physical activity was assessed by using a 12-month leisure-time history based on self-reported information about frequency per month over the preceding year, average duration per occasion, and intensity level. Metabolic units were assigned for each activity according to intensity. Physical activity was expressed as kcal/d.¹⁹ Education, symptomatic CHD or CHD history, diabetes, medication, use of supplements and smoking were collected with a self-administered questionnaire and checked by the interviewer. Education was coded into three categories based on years of education (<6, 7–11 and 12 or more years).²⁰ A subject was defined a smoker if he had ever smoked on a regular basis and had smoked cigarettes, cigars or a pipe within the past 30 days. The lifelong exposure to smoking was estimated as the product of the number of smoking years and the number of tobacco products smoked daily at the time of examination.²¹

Collection and classification of AMI

Data on AMI were obtained from the National Hospital Discharge Data Register by record linkage. The diagnostic classification of coronary events was based on symptoms, electrocardiographic findings and cardiac enzyme elevations. Each suspected coronary event was coded according to the Ninth (code numbers 410–414) or Tenth (code numbers I20–I25) International Classification of Diseases (ICD) and was classified into (i) a definite AMI, (ii) a probable AMI, (iii) a typical acute chest pain episode of more than 20 min indicating CHD, (iv) an ischaemic cardiac arrest with successful resuscitation or (v) no acute coronary event by a physician using the original patient records. All AMI cases that occurred from the study entry (i.e. 1991) until 31 December 2005 were included. If a participant had multiple events, the first event was considered as the end point.

Statistical methods

Descriptive data were presented as means, standard deviations (SD) and percentages. The independent-samples t-test was used to examine the differences in the concentrations of serum carotenoids and other covariates between AMI cases and the other men. Correlations between carotenoids and risk factors were assessed by using Pearson's correlation coefficients. Subjects were classified into tertiles according to their serum concentrations of carotenoids. Tests for linear trend were calculated by using means option. The risk ratios (RR) and 95% confidence intervals (CI) for AMI in tertiles of serum concentrations of carotenoids were estimated by using Cox proportional hazard model. Two different sets of covariates were used: (i) age and examination year; (ii) age, examination year BMI, systolic blood pressure, smoking (yes vs. no), alcohol consumption, serum LDL cholesterol, years of education, symptomatic CHD or CHD history, diabetes, physical activity, antihypertensive medication, drug for high cholesterol and any β-adrenergic blocking agent. Covariates were selected on the basis of their previously established role as a predictive factor on the basis of overall evidence and available data. The cumulative survival curves from AMI according to tertiles of serum lycopene and β-carotene concentrations were calculated using the Cox proportional hazards’ method adjusting for covariates. Tests for statistical significance were two sided and differences with P < 0.05 were considered statistically significant. SPSS software (version 14.0; SPSS, Inc., Chicago, IL, USA) was used for statistical analyses.

Results

The present study included a total of 194 incidents AMI cases occurred during an average of 11.5 follow-up years (range 0.03–14.8 years). Men who developed AMI were older, had lower education, performed less physical activity, were more likely to have symptomatic CHD or CHD history, diabetes and to smoke cigarettes (pack-year), but they consumed less alcohol. AMI patients had also higher systolic blood pressure, concentrations of serum LDL cholesterol, triglycerides and α-tocopherol, but lower concentrations of serum HDL cholesterol and serum lycopene. Patients with AMI used more antihypertensive drugs and β-adrenergic blocking agents. Other characteristics were similar to those in other men (table 1). Serum α-carotene and β-carotene concentrations were significantly (P < 0.05) lower in smokers than non-smokers (α-carotene: 0.077 vs. 0.10 µmol/l and β-carotene: 0.33 vs. 0.40 µmol/l). However, smoking was not found to be a significant interaction term.

Table 1

Demographic characteristics of the study population with and without AMI (n = 1031 men)

	Men without AMI (n = 844)	Men with AMI (n = 194)	P-value
Demographic
Age (years)	55.7 (6.7)a	58.5 (6.2)	<0.0001b
BMI (kg/m2)	27.5 (3.6)	27.8 (3.6)	0.213
Years of education	9.4 (3.7)	8.7 (2.9)	0.014
Physical activity (kcal/d)	175.7 (210.9)	176.4 (204.7)	0.967
Medical history
Systolic blood pressure (mm Hg)	135 (16)	139 (18)	0.003
Diastolic blood pressure (mm Hg)	89 (10)	90 (11)	0.339
Symptomatic CHD or CHD history (%)	17.0c	41.0	<0.0001d
Diabetes (%)	6.0	11.0	<0.0001
Alcohol consumption (g/week)	81.0 (125.9)	73.5 (125.3)	0.455
Smokers (%)	27.0	30.0	<0.0001
Smoking (pack-years)	5.2 (11.4)	7.2 (14.6)	0.048
Medications
Drug for hypertension (%)	24.0	48.0	<0.0001
Any β-adrenergic blocking agent (%)	9.0	22.0	<0.0001
Laboratory data
Serum retinol (µmol/l)	2.11 (0.45)	2.10 (0.46)	0.689
Serum α-tocopherol (µmol/l)	28.3 (7.6)	30.4 (9.3)	0.001
Serum lycopene (µmol/l)	0.16 (0.14)	0.11 (0.11)	<0.0001
Serum α-carotene (µmol/l)	0.097 (0.078)	0.093 (0.081)	0.540
Serum β-carotene (µmol/l)	0.39 (0.30)	0.36 (0.28)	0.157
Serum HDL cholesterol (mmol/l)	1.11 (0.28)	1.04 (0.30)	0.004
Serum LDL cholesterol (mmol/l)	3.89 (0.83)	4.12 (0.87)	0.001
Serum triglycerides (mmol/l)	1.57 (1.00)	1.84 (1.15)	0.001

Bold values are statistically significant.

a: Continuous variables are presented as mean (standard deviations).

b: P for differences between those with and without AMI for continuous variables (the independent-samples t-test).

c: Dichotomous variables are presented as percentage.

d: P-value for categorical variables (chi-squared test).

Pearson's correlation coefficients were calculated to assess the relation between carotenoids and possible risk factors for AMI/myocardial infarction-associated risk factors. All carotenoids were inversely (P < 0.05) correlated with BMI, systolic blood pressure, diastolic blood pressure, smoking (pack-year), drug for hypertension, symptomatic CHD or CHD history and use of any β-adrenergic blocking agents. In addition, lycopene correlated inversely with age (r = −0.30). All carotenoids correlated with years of education (r = 0.06 − 0.33). Carotenoids were strongly correlated with each other. For α-carotene and β-carotene r = 0.61 (P < 0.001), for α-carotene and lycopene r = 0.16 (P < 0.001) and for lycopene and β-carotene r = 0.25 (P < 0.001).

We observed significant linear trends towards a higher risk of AMI across tertiles of serum concentrations of lycopene and β-carotene (P-value for trend <0.001 and 0.013, respectively). The association between tertiles of serum lycopene and β-carotene and the risk of AMI are presented in table 2. After adjustment for age and examination year, low serum lycopene and β-carotene increased the risk of AMI. RR, 1.68 (95% CI 1.16–2.43; P = 0.006) for lycopene and RR, 1.75 (95% CI 1.22–2.50; P = 0.002) for β-carotene. After further adjusting for BMI, SBP, smoking, alcohol intake, serum LDL cholesterol, years of education, symptomatic CHD or CHD history, diabetes, physical activity, antihypertensive medication, drug for high cholesterol and any β-adrenergic blocking agent, men in the lowest tertiles of serum concentrations of lycopene and β-carotene increased the risk of AMI by 55 and 60% as compared to those in the highest tertiles RR, 1.55 (95% CI 1.05–2.30; P = 0.028) for lycopene and RR, 1.60 (95% CI 1.09–2.35; P = 0.017) for β-carotene. The cumulative survival curves according to the tertiles of serum lycopene and β-carotene concentrations continued to diverge during the follow-up period. Serum concentrations of α-carotene, α-tocopherol and retinol were not associated with the risk of AMI.

Table 2

Relative risks and 95% confidence intervals of acute myocardial infarction by tertiles of serum concentrations of carotenoids and fat-soluble vitamins

	Tertiles of carotenoids and fat-soluble vitaminsa
	1 (highest)	2	3 (lowest)	P-value
Model 1: Age and examination year adjustedb
Lycopene	1.0	1.06 (0.72–1.57)	1.68 (1.16–2.43)	0.006
α-Carotene	1.0	1.04 (0.74–1.47)	1.14 (0.80–1.64)	0.471
β-Carotene	1.0	1.37 (0.95–1.97)	1.75 (1.22–2.50)	0.002
Retinol	1.0	1.0 (0.70–1.43)	1.06 (0.75–1.51)	0.737
α-Tocopherol	1.0	0.73 (0.52–1.02)	1.61 (0.43–0.86)	0.005
Model 2: Multivariate adjustedc
Lycopene	1.0	1.09 (0.73–1.62)	1.55 (1.05–2.30)	0.028
α-Carotene	1.0	0.93 (0.66–1.32)	0.98 (0.67–1.42)	0.900
β-Carotene	1.0	1.24 (0.85–1.80)	1.60 (1.09–2.35)	0.017
Retinol	1.0	1.13 (0.79–1.61)	1.16 (0.81–1.67)	0.413
α-Tocopherol	1.0	0.89 (0.63–1.27)	0.75 (0.51–1.12)	0.160

a: Tertiles of carotenoids and fat-soluble vitamins (µmol/l): lycopene: <0.08, 0.08–0.19, >0.19; α-Carotene: <0.06, 0.06–0.11, >0.11; β-Carotene: <0.25, 0.25–0.40, >0.40; Retinol: <1.88, 1.88–2.25, >2.25; α-Tocopherol: <24.6, 24.6–30.3, >30.3.

b: Adjusted for age and examination year.

c: Adjusted for age, examination year, BMI, SBP, smoking, alcohol intake, serum LDL cholesterol, years of education, physical activity, symptomatic CHD or CHD history, diabetes, antihypertensive medication, drug for high cholesterol and any β-adrenergic blocking agent.

Discussion

In this population-based prospective study, our primary finding was that low serum concentrations of lycopene and β-carotene may increase the risk of AMI in men. Conversely, there was no statistically significant association between serum levels of α-carotene, or any other fat-soluble vitamins and risk of AMI. Cumulative survival curves (figures 1 and 2) refer to AMI survivors. Group 1 (the lowest tertile of concentration) describes the highest risk of AMI and Group 3 (the highest tertile of concentration) the lowest risk of AMI, respectively.

Figure 1

Cumulative survival curves of AMI according to the tertiles of serum lycopene concentrations during follow-up period. The cut-offs were <0.08 µmol/l (1), 0.08–0.19 µmol/l (2) and >0.19 µmol/l (3)

Figure 2

Cumulative survival curves of AMI according to the tertiles of serum β-carotene concentrations during follow-up period. The cut-offs were <0.25 µmol/l (1), 0.25–0.40 µmol/l (2) and >0.40 µmol/l (3)

Our results are consistent with some epidemiological findings, which support a protective role of lycopene and/or β-carotene on the risk of AMI. Studies have suggested that high plasma concentrations of lycopene and β-carotene tend to be associated with lower risk of MI.^7,10,12 In some previous studies, high dietary intake of β-carotene was shown to decrease the risk of AMI.^8,9 In an Italian population, a weak protective effect of dietary α-carotene, β-carotene and β-cryptoxanthin, but not lycopene, lutein or zeaxanthin, on the risk of AMI was observed.²³ The Singapore Chinese Health Study demonstrated an inverse association between plasma levels of β-cryptoxanthin and lutein and the risk of developing AMI.¹¹ In addition, adipose tissue concentrations of carotenoids are inversely related to MI.^6,9,13 However, some recent studies do not support the protective role of plasma carotenoids against AMI.^5,14,15

Seasonal variations in the concentrations of carotenoids were observed. Lycopene concentrations tended to be highest between July and September, when tomato consumption is usually most abundant. Conversely, consumption of carrots seems to be highest in the late autumn and winter, reflecting the highest concentrations of α-carotene and β-carotene in the blood. Serum concentrations at the end of follow-up were available, but they were not comparable with baseline because of another HPLC method used²² (data not shown).

Lycopene concentration was 59% lower, α-carotene 17% and β-carotene 7% higher at the end of the follow-up as compared with baseline levels. We cannot exclude the possibility that changes of carotenoid levels from baseline to the end of the follow-up are partly due to analytical changes. Also dietary changes may be possible. At the end of follow-up visit, the sample type was Li-heparin plasma, whereas at the baseline visit collected samples were serum. It has been reported that no significant differences in the concentrations of carotenoids can be found between samples measured from serum or Li-heparin plasma, although these concentrations were slightly higher in serum.²⁴ Peak-height was used in our previous and current method for quantification of carotenoids. We observed that total lycopene (cis + trans) was not measured at the baseline. Therefore, difference in the levels of lycopene between methods was not significant under in vivo circumstances.

Oxidative modification of LDL in the vascular endothelium is suggested to initiate the development of early atherosclerosis.^25,26 Under oxidative stress by reactive oxygen species, polyunsaturated fatty acids of LDL are easily oxidized producing a number of oxidation products (e.g. conjugated dienes, C₁₈ hydroxy fatty acids, malondialdehyde).^27,28 Atherosclerosis is a pathological condition in which arteries undergo thickening of their intimal regions and lose their elasticity. However, atherosclerosis itself is rather asymptomatic and early changes in vessel walls that lead to atherosclerotic lesions that are found even in healthy young people. It is well known that the coronary arteries, cerebral arteries and the aorta are the major sites of atherosclerosis. Extensive atherosclerotic development narrows the lumen of the arteries and reduces blood flow.²⁸

It has been shown that the plasma levels of oxidative modified LDL (ox-LDL) are higher in patients with AMI.²⁹ In addition, increased levels of oxidized LDL were observed in patients with CHD.²⁹ A recent in vivo study has provided evidence that ox-LDL can be used to predict future atherosclerotic events.³⁰ It has been suggested that high ox-LDL levels in the acute phase of AMI could be caused by a massive release of ox-LDL from the ruptured plaques, whereas high levels during the stable phase may indicate that the patients have strong sources of ox-LDL production. Such sources may include exposure to strong oxidative stress or the presence of unstable plaques somewhere in blood vessels that release ox-LDL from the lesions.²⁸

Carotenoids are most likely to be involved in the scavenging of two reactive oxygen species, singlet molecular oxygen and peroxyl radicals. β-Carotene, zeaxanthin β-cryptoxanthin and α-carotene belong to the group of highly active quenchers of singlet oxygen.³¹ The most efficient carotenoid is the open ring carotenoid lycopene.³² Carotenoids scavenge peroxyl radicals generated in the process of lipid oxidation interrupts the reaction sequence that finally leads to damage in lipophilic compartments (e.g. ox-LDL) and to initiation and progression of atherosclerosis.³ There are numerous intervention studies concerning supplementation of healthy subjects with food stuffs (e.g. tomato products), in which effects of carotenoids on lipid oxidation are evaluated.^33–38 However, most of the previous dietary supplementation studies have been uncontrolled leading to quite uncertain results. It seems that most of intervention studies do not support the protective role of carotenoid supplementation against lipid oxidation.^33,37–39

Since individuals consuming diets high in fruits and vegetables tend to have high concentration of carotenoids, the strong association between lycopene and β-carotene may conceivably explain the association between β-carotene and AMI evident in some observational studies. On the basis of our study findings, β-carotene may be a good marker of lycopene intake, and its effect disappears, when both are considered simultaneously. In the same line of argument, lycopene could be a good marker of other active phytochemicals in tomatoes, and possibly in other major vegetable sources of this carotenoid.⁶ In addition, combinations of different antioxidants, as found in fruits and vegetables, may have the pronounced health benefits. In a previous study, the combination of phenolic compounds and carotenoids led to synergistic effects by preventing oxidation of human LDL more effectively than carotenoids alone.⁴⁰

The strengths of this study include its prospective population-based design, complete follow-up (no losses), and reliable assessments of incident AMIs. Since blood samples were collected at baseline when all men were AMI-free, these concentrations do not represent any changes made after diagnosis. Therefore it is impossible to know, if patients had changed their lifestyles and dietary habits by increasing intake of dietary antioxidants after AMI diagnosis. This is also difficult to estimate because of different HPLC method used at the end of follow up. However, it may be possible that seasonal variations in concentrations of lycopene and β-carotene have affected on results.

This study demonstrated that low serum lycopene and β-carotene concentrations may increase the risk of AMI in men. The results of this study support that the intake of foods rich in lycopene and β-carotene may be considered to be useful in protecting from AMI.

Key points

• Low serum lycopene and β-carotene concentrations may increase the risk of AMI in men.

• Healthy vegetable rich food may be useful protecting against cardiovascular diseases and AMI and increase public health in middle-aged men.

• Our results support beneficial effects of carotenoids against AMI as it has been described lot of in the literature.

Acknowledgements

The authors acknowledge the staff of the Institute of Public Health and Clinical Nutrition at the University of Eastern Finland for helping with data collection. The authors thank to Kimmo Ronkainen for help in the statistical analyses.

Conflicts of interest: None declared.