This study provides evidence that higher beta carotene biochemical status is associated with lower overall, cardiovascular disease, heart disease, stroke, cancer, and other causes of mortality. The dose-response associations over a 30-year period were not attenuated by adjustment for other important risk factors and support greater fruit and vegetable consumption as a means to increase beta carotene status and promote longevity.

We conducted a prospective serological analysis of 29 103 men in the ATBC study (Alpha-Tocopherol, Beta-Carotene Cancer Prevention). During 31 years of follow-up, 23 796 deaths occurred, including deaths because of cardiovascular disease (9869), cancer (7692), respiratory disease (2161), diabetes mellitus (119), injuries and accidents (1255), and other causes (2700). Serum beta carotene was assayed using high-performance liquid chromatography. Adjusting for major risk factors measured, men with higher serum beta carotene had significantly lower all-cause mortality (hazard ratios=0.81, 0.71, 0.69, and 0.64 for quintile 2 (Q2)-Q5 versus Q1, respectively; P trend <0.0001). Serum beta carotene was significantly associated with risk of death from cardiovascular disease, heart disease, stroke, cancer, respiratory disease, diabetes mellitus, injuries and accidents, and other causes (Q5 versus Q1, hazard ratio=0.21–0.73, all P trend <0.0001). The all-cause mortality association was not materially impacted by adjustment for fruit and vegetable consumption (albeit, estimated with some measurement error) and was generally similar across subgroups of smoking intensity, alcohol consumption, trial supplementation, and duration of follow-up, but was significantly modified by age, years of smoking, and body mass index, with stronger inverse associations among men who were younger, smoked fewer years, and had a lower body mass index (all P interaction ≤0.0025).

Explore whether serum beta carotene is associated with overall and cause-specific mortality and to elucidate the strength and dose-response of the association.

Although the health effects of beta carotene have been studied extensively, a systematic examination of serum concentrations and long-term mortality, including cardiovascular disease mortality, has not been reported.

Despite substantial attention focused on the human health effects of beta carotene, a systematic examination of the association between serum concentrations and long-term mortality has not been reported. This is particularly true with respect to cause-specific mortality (eg, cardiovascular disease [CVD]) and the examination of dose-response relationships. The phytochemical beta carotene is found naturally in green leafy vegetables, fruits, and other yellow/orange plants, and it can be synthesized by microorganisms.1 As the major carotenoid, beta carotene can be metabolized into bioactive retinol and other beta carotene compounds, which are critical for maintaining normal human physiology and homeostasis.2 In addition, previous in vitro and in vivo studies demonstrate that beta carotene is a powerful antioxidant capable of neutralizing intracellular free radicals involved in the development of chronic illnesses, including CVD and cancer. Serum beta carotene has also been inversely associated with systemic markers of inflammation and insulin resistance.3,4

Editorial, see p 1267

In This Issue, see p 1255

Meet the First Author, see p 1256

Several studies, including a recent meta-analysis, suggest an inverse association between circulating beta carotene and total mortality,5–9 although there have been mixed results for risk of death from cancer and CVD, and data are sparse for heart disease, stroke, respiratory disease, and diabetes mellitus mortality.6–14 Most of these studies have been based on relatively small numbers of deaths and had limited power to examine cause-specific mortality, dose-response associations, and risk among population subgroups. At the same time, large controlled trials reported either no benefits or small unexpected adverse effects of supra-physiological beta carotene supplementation, including increased overall mortality and cancer incidence.15–18 A better understanding of the beta carotene-mortality association, and test of dose-response, is clearly needed.

The present study examined the association between serum beta carotene and risk of mortality from all causes, total CVD, heart disease, stroke, cancer, respiratory disease, diabetes mellitus, injuries/accidents, and other causes in a large prospective cohort of men with 30 years of follow-up and nearly 24 000 deaths.

Methods

Because of cohort data use agreements in place between the United States and Finland, the data, analytic methods, and study materials will not be made available to other researchers for the aim of reproducing the results.

Study Population

The ATBC study (Alpha-Tocopherol, Beta-Carotene Cancer Prevention) has been described in detail.19 The study was a 2×2 factorial, randomized, double-blind, placebo-controlled primary prevention trial that examined whether alpha-tocopherol and beta carotene reduced the incidence of cancer.19 Briefly, 29 133 male smokers from southwest Finland, aged 50 to 69 years, were eligible and randomly assigned to receive 1 of 4 supplements daily (alpha-tocopherol [50 mg], beta carotene [20 mg], both or placebo) for 5 to 8 years between 1985 and 1988 until the end of the intervention (April 30, 1993). Self-administered questionnaires were completed at enrollment, including information about general health, lifestyle risk factors, and a validated food frequency questionnaire, and height, weight, blood pressure, and heart rate were measured. Presupplementation fasting blood samples were collected and stored at −70°C until biochemically assayed. Written informed consent was provided by all participants, and the Study was approved by the Institutional Review Boards at the Finnish National Public Health Institute and the US National Cancer Institute.

Serum concentrations of beta carotene, alpha-tocopherol, and retinol were assayed using high-performance liquid chromatography,19,20 and the coefficient of variation for serum beta carotene was 6.5%.20 After excluding participants without a serum beta carotene measurement (N=30), 29 103 were included in the final analysis (from a total of 29 133 participants in the ATBC parent trial cohort).

Outcome Assessment

Specific causes of death from baseline through the end of the follow-up (December 31, 2015) were ascertained via linkage with the Causes of Death Registry, Statistics Finland. We used the following codes from the Eighth, Ninth, and Tenth revisions of International Classification of Diseases (ICD-8, -9, -10, respectively) to classify the underlying cause of death: CVD (ICD-8, 390–458; ICD-9, 390–459; and ICD-10, I00-I99), heart disease (ICD-8 and ICD-9, 390–398, 401–404, 410–429, and 440–448; ICD-10, I00-I13, I20-I51, and I70-I78), stroke (ICD-8 and ICD-9, 430–438 and ICD-10, I60-I69), cancer (ICD-8 and ICD-9, 140–239; ICD-10, C00-D48), respiratory disease (ie, pneumonia, influenza, chronic obstructive pulmonary disease, and other related conditions; ICD-8, 470–474, 480–486, 490–493, and 518; ICD-9, 480–487 and 490–496; and ICD-10, J10-J18 and J40-J47), diabetes mellitus (ICD-8 and ICD-9, 250 and ICD-10, E10-E14), injuries and accidents (ICD-8, 800–978; ICD-9, E800-E978; and ICD-10, V01-X59, Y85-Y86, U03, X60-X84, Y87.0, Y87.1, U01-U02, X85-Y09, Y35, and Y89.0), and all other causes combined.

Statistical Analysis

We calculated person-time of follow-up for each participant from the date of randomization to the date of death or the end of follow-up (December 31, 2015), whichever occurred first. We used Cox proportional hazards regression models with attained age as the time metric to estimate the hazard ratio (HR) and 95% confidence interval (CI) for associations between serum beta carotene (quintiles) and mortality risk, including overall and cause-specific mortality. In cause-specific models, mortality other than the event of interest was censored at the time of death. The proportional hazards assumption was not violated based on inspection of the Schoenfeld residuals of exposure variables of interest. Tests for linear trend were constructed by assigning the median value of each quintile and entering it to the model as a continuous variable. The crude model was adjusted for age at blood collection (continuous variable). Multivariate models were additionally adjusted for number of cigarettes smoked per day, number of years of smoking, trial intervention group (beta carotene or no beta carotene, alpha-tocopherol or no alpha-tocopherol), systolic and diastolic blood pressure, serum total cholesterol, and HDL (high-density lipoprotein) cholesterol. In the models with CVD, heart disease, and stroke as endpoints, we further adjusted for history of CVD. In addition, the following covariates were assessed but were not included in the final model as none altered the estimated main effect of serum beta carotene by 10% or more: body mass index (BMI, kg/m2), physical activity, baseline beta carotene supplement use, history of diabetes mellitus, energy intake, educational status, and daily intakes of alpha-carotene, fruit, vegetables, fat, red meat, alcohol, total fiber, and cereals.

We constructed Kaplan-Meier survival curves according to serum beta carotene quintiles and compared differences for the composite endpoints across groups using the log-rank test. We used cubic restricted splines with 4-knots to account for possible non-linear associations between serum beta carotene concentrations and mortality risk. Knots were selected at the fifth, 25th, 75th, and 95th percentiles of the serum beta carotene concentration.

We performed stratified analyses by age at baseline (<54, 54–59, or ≥59 years), number of cigarettes smoked per day (<16, 16–20, or >20), number of years of smoking (<33, 33–40, or ≥40 years), daily alcohol consumption (<5.3, 5.3–20.4, or ≥20.4 g), BMI (<25, 25–28, or ≥28 kg/m2), intervention group assignment (beta carotene or no beta carotene, alpha-tocopherol or no alpha-tocopherol), and years of follow-up (<13, 13–23, or ≥23 years). P values for interactions were evaluated through likelihood ratio tests by comparing Cox proportional hazards models with and without the cross-product terms for each assessed factors and baseline serum beta carotene (quintile). We conducted 8 interaction tests for each outcome, therefore, differences with P<0.05 could be considered chance findings. To minimize the reverse causality bias, we performed a sensitivity analysis that excluded the first 5 years of follow-up. To decrease the potential bias from effects of preexisting illness on serum beta carotene concentration, we performed sensitivity analyses that excluded participants who reported a history of CVD, diabetes mellitus, or both at baseline.

All analyses were performed using SAS software (version 9.4; SAS Institute Inc, Cary, NC), and statistical tests and reported P values were 2-tailed.

Results

Mean baseline serum beta carotene concentration in the cohort was 212 µg/L, and mean beta carotene in the fifth quintile was nearly 7× higher than that in the first quintile (Table 1). As compared with men in the lowest quintile, subjects in the higher quintiles of beta carotene smoked less, were more physically active and likely to use vitamin supplements, and less likely to have a history of CVD or diabetes mellitus. Serum beta carotene was positively associated with fruit and vegetable consumption, alpha-carotene and fiber intake, and serum total cholesterol (the latter likely because of beta carotene transport in lipoproteins; Table 1).

Table 1. Baseline Population Characteristics by Quintile of Serum Beta Carotene in the ATBC Study* Quintile of Serum Beta Carotene Quintile 1 (n=5797) Quintile 2 (n=5731) Quintile 3 (n=5891) Quintile 4 (n=5815) Quintile 5 (n=5869) Serum beta carotene, µg/L 66.8 (21.8) 120.9 (13.6) 170.5 (15.8) 238.8 (25.4) 457.8 (273.0) Age, y 57.0 (4.9) 57.3 (5.1) 57.3 (5.1) 57.2 (5.1) 57.3 (5.1) Cigarettes/d 22.2 (9.3) 20.9 (8.8) 20.4 (8.8) 19.8 (8.6) 18.8 (8.3) Years smoked, y 36.4 (8.1) 36.3 (8.4) 35.9 (8.5) 35.6 (8.7) 35.4 (8.7) Systolic blood pressure, mm Hg 146.5 (19.8) 144.0 (19.4) 141.4 (19.0) 139.7 (19.1) 138.3 (18.9) Diastolic blood pressure, mm Hg 90.5 (11.1) 88.7 (10.7) 87.4 (10.5) 86.2 (10.5) 85.2 (10.5) Serum total cholesterol, mmol/L 5.8 (1.2) 6.1 (1.1) 6.2 (1.1) 6.4 (1.1) 6.6 (1.2) Serum HDL cholesterol, mmol/L 1.26 (0.39) 1.18 (0.33) 1.18 (0.30) 1.17 (0.28) 1.19 (0.28) Serum alpha-tocopherol, mg/L 10.9 (4.6) 11.6 (3.4) 11.9 (3.1) 12.2 (2.9) 12.9 (3.2) BMI, kg/m2 26.8 (4.3) 26.9 (3.9) 26.4 (3.7) 26.0 (3.6) 25.4 (3.3) Physically active, % 15.0 19.3 20.8 23.2 25.5 History of CVD,† % 46.8 44.3 40.6 39.2 36.6 History of diabetes mellitus, % 7.2 4.9 3.6 2.9 2.7 Beta carotene supplement use, % 8.0 9.1 9.0 10.0 15.6 Vitamin E supplement use, % 8.0 9.3 9.0 9.9 14.5 Daily dietary intake Energy, kcal 2617 (768) 2676 (761) 2716 (739) 2719 (750) 2714 (746) Alcohol (g ethanol) 30.0 (28.0) 20.5 (22.1) 16.4 (19.0) 13.2 (16.6) 10.5 (14.4) Fruit, g 106 (93.0) 122 (97.5) 128 (99.1) 137 (104.8) 150 (109.3) Vegetables, g 96.3 (61.4) 106.7 (65.2) 111.2 (68.0) 118.6 (71.8) 134.1 (79.9) Fat (triacylglycerol), g 98.8 (35.8) 105 (35.8) 107 (35.4) 108 (36.2) 109 (37.0) Red meat, g 68.3 (34.1) 70.9 (33.3) 72.0 (34.3) 72.4 (33.3) 72.8 (34.3) Alpha-carotene, µg 458 (452) 541 (501) 596 (549) 668 (597) 855 (771) Total fiber, g 17.0 (9.8) 18.2 (9.9) 19.0 (10.0) 19.5 (10.0) 20.0 (10.0) Total cereal, g 194 (85) 211 (85) 212 (86) 224 (86) 229 (86) Education (%, > elementary school) 21.0 21.3 19.6 20.2 23.1

During 31 years of follow-up (median, 18 years and total person-years, 514 271), 23 796 men died (81.8%), including 9869 deaths from CVD disease (8064 heart disease deaths and 1764 stroke deaths), 7692 deaths from cancer, 2161 deaths from respiratory disease, 119 deaths from diabetes mellitus, 1255 deaths from injuries and accidents, and 2700 deaths from all other causes. Compared with men in the lowest quintile of serum beta carotene, those in the higher quintiles experienced significantly lower age-adjusted mortality from all causes, CVD, heart disease, stroke, cancer, respiratory disease, diabetes mellitus, injuries and accidents, and all other causes, representing 34% to 83% risk reductions (all P trend <0.0001; Table 2). After multivariate adjustment, the inverse associations remain statistically significant with the HRs attenuated slightly to 27% to 79% lower risk in the highest beta carotene quintile (all P trend <0.0001; Table 2). Further adjustment for BMI, intakes of alcohol, alpha-carotene, fruits and vegetables, total fiber and cereal, and educational status, also did not change the association estimates appreciably (Online Tables I through III). Kaplan-Meier survival plots demonstrate that men in the lowest quintile of serum beta carotene had significantly increased cumulative overall mortality than those in the higher quintiles (log-rank test P value<0.0001; Figure 1A), and similar patterns were observed for cause-specific mortality (all log-rank test P values<0.0001; Figure 1B through 1D; Online Figure I).

Table 2. HRs and 95% CI for All-Cause and Cause-Specific Mortality by Quintile of Serum Beta Carotene in the ATBC Study* Serum Beta Carotene Quintile 1 Quintile 2 Quintile 3 Quintile 4 Quintile 5 All-cause mortality Deaths, n 5193 4857 4728 4568 4450 Death rate† 58.2 49.9 44.3 42.3 39.4 Age-adjusted HR (95% CI) 1.0 0.78 (0.75–0.82) 0.68 (0.65–0.70) 0.64 (0.61–0.66) 0.57 (0.55–0.60) Multivariate HR (95% CI)‡ 1.0 0.81 (0.78–0.84) 0.71 (0.69–0.74) 0.69 (0.66–0.72) 0.64 (0.61–0.67) CVD mortality Deaths, n 2085 2052 2041 1890 1801 Death rate† 23.36 21.07 19.14 17.51 15.94 Age-adjusted HR (95% CI) 1.0 0.83 (0.78–0.88) 0.73 (0.69–0.78) 0.66 (0.62–0.71) 0.59 (0.55–0.62) Multivariate HR (95% CI) ‡§ 1.0 0.84 (0.79–0.89) 0.76 (0.72–0.81) 0.71 (0.66–0.75) 0.64 (0.60–0.68) Heart disease Deaths, n 1681 1695 1669 1536 1483 Death rate† 18.83 17.40 15.65 14.23 13.12 Age-adjusted HR (95% CI) 1.0 0.85 (0.80–0.91) 0.75 (0.70–0.80) 0.67 (0.63–0.72) 0.60 (0.56–0.65) Multivariate HR (95% CI)‡§ 1.0 0.85 (0.79–0.91) 0.76 (0.71–0.82) 0.70 (0.65–0.75) 0.64 (0.59–0.68) Stroke Deaths, n 395 347 364 342 316 Death rate† 4.43 3.56 3.41 3.17 2.80 Age-adjusted HR (95% CI) 1.0 0.72 (0.62–0.83) 0.67 (0.58–0.77) 0.61 (0.52–0.70) 0.52 (0.45–0.60) Multivariate HR (95% CI)‡§ 1.0 0.78 (0.68–0.91) 0.78 (0.67–0.90) 0.74 (0.64–0.86) 0.67 (0.57–0.78) Cancer mortality Deaths, n 1584 1543 1501 1546 1518 Death rate† 17.75 15.84 14.07 14.32 13.43 Age-adjusted HR (95% CI) 1.0 0.83 (0.77–0.89) 0.72 (0.67–0.77) 0.72 (0.67–0.77) 0.66 (0.62–0.71) Multivariate HR (95% CI)‡ 1.0 0.85 (0.80–0.92) 0.75 (0.70–0.81) 0.78 (0.72–0.84) 0.73 (0.68–0.79) Respiratory disease Deaths, n 499 434 404 412 412 Death rate† 5.59 4.46 3.79 3.82 3.65 Age-adjusted HR (95% CI) 1.0 0.69 (0.61–0.79) 0.56 (0.49–0.64) 0.55 (0.48–0.63) 0.51 (0.44–0.58) Multivariate HR (95% CI)‡ 1.0 0.81 (0.71–0.92) 0.69 (0.60–0.79) 0.72 (0.63–0.83) 0.71 (0.62–0.82) Diabetes mellitus Deaths, n 41 28 20 19 11 Death rate† 0.46 0.29 0.19 0.18 0.10 Age-adjusted HR (95% CI) 1.0 0.56 (0.35–0.91) 0.35 (0.21–0.60) 0.32 (0.19–0.56) 0.17 (0.09–0.34) Multivariate HR (95% CI)‡ 1.0 0.57 (0.35–0.93) 0.37 (0.22–0.65) 0.37 (0.21–0.64) 0.21 (0.11–0.42) Injuries and accidents Deaths, n 301 286 254 222 192 Death rate† 3.37 2.94 2.38 2.06 1.70 Age-adjusted HR (95% CI) 1.0 0.84 (0.71–0.99) 0.69 (0.58–0.81) 0.59 (0.49–0.70) 0.48 (0.40–0.57) Multivariate HR (95% CI)‡ 1.0 0.93 (0.79–1.10) 0.78 (0.66–0.93) 0.69 (0.58–0.83) 0.57 (0.47–0.69) Other causes Deaths, n 683 514 508 479 516 Death rate† 7.65 5.28 4.76 4.44 4.57 Age-adjusted HR (95% CI) 1.0 0.61 (0.54–0.68) 0.51 (0.46–0.58) 0.47 (0.42–0.53) 0.46 (0.41–0.51) Multivariate HR (95% CI)‡ 1.0 0.64 (0.57–0.72) 0.55 (0.49–0.62) 0.51 (0.45–0.58) 0.51 (0.45–0.58)

Figure 1. Kaplan-Meier plots of overall and cause-specific mortality by quintiles of serum beta carotene in the ATBC study (Alpha-Tocopherol, Beta Carotene Cancer Prevention). A, Overall mortality. B, Cardiovascular disease (CVD) mortality. C, Heart disease mortality. D, Stroke mortality.

To evaluate the serum beta carotene-mortality dose-response relation, we used a restricted 4-knot cubic spline regression, modeling serum beta carotene as a continuous variable. All-cause and cause-specific relative mortality increased as serum beta carotene values decreased below the reference value of 98 µg/L (corresponds to the cutoff value of the first quintile), whereas significant reductions in risk were observed for increasing serum beta carotene concentrations, with association plateaus suggested for CVD, heart disease, and stroke mortality (Figure 2; Online Figure II).

Figure 2. Cubic spline regression for estimated relative risk of overall and cause-specific death according to serum beta carotene concentrations in the ATBC study (Alpha-Tocopherol, Beta-Carotene Cancer Prevention). The reference value (98 µg/L; relative risk=1) corresponds to the cutoff value of the first quintile of serum beta carotene concentration. A, Overall mortality. B, Cardiovascular disease (CVD) mortality. C, Heart disease mortality. D, Stroke mortality. The solid line indicates the relative risk for mortality and serum beta carotene with a 4-knot spline (knots were selected at the fifth, 25th, 75th, and 95th percentiles of the serum beta carotene); dashed lines indicated the 95% confidence intervals. Total number of participants: 29 103. Event number of overall, CVD, heart disease, and stroke mortality is 23 796, 9869, 8064, and 1764, respectively.

Evaluating effect modification of the all-cause mortality association by baseline factors and duration of follow-up revealed generally similar findings for serum beta carotene across subgroups of cigarettes smoked daily, alcohol consumption, trial intervention, and duration of follow-up. The beta carotene association was, however, statistically significantly modified by baseline age (P interaction <0.0001), cumulative years of smoking (P interaction =0.0025), and BMI (P interaction =0.0009; Table 3) with stronger inverse associations for men <54 years old, those with <33 years of smoking, and those with BMI <25 kg/m2, the latter 2 findings being independent of age, based on the multivariable-adjusted models. Associations between beta carotene concentration and cause-specific mortality were also generally similar across categories of duration of follow-up (except for other causes of mortality), alcohol consumption, and trial intervention group (Online Tables IV through VII). The inverse association with CVD and heart disease mortality was stronger only among younger men (P interaction <0.0001 and P interaction =0.0012, respectively; Online Tables IV and V), whereas the inverse association with stroke and cancer mortality appeared stronger in those with <20 cigarettes smoked daily and those with BMI <25 kg/m2, respectively (Online Tables VI and VII, respectively). There were no significant subgroup differences for respiratory disease, injury/accidental deaths, or other causes, other than a strong inverse association in the first 13 years follow-up for other causes (data not shown).

Table 3. HRs and 95% CI for All-Cause Mortality by Quintile of Serum Beta Carotene in the ATBC Study, by Baseline Subgroups* Total No. of Subjects Serum Beta Carotene P Value for Interaction‡ Quintile 1 Quintile 2 Quintile 3 Quintile 4 Quintile 5 Events/Death Rate† HR Events/Death Rate† HR (95% CI) Events/Death Rate† HR (95% CI) Events/Death Rate† HR (95% CI) Events/Death Rate† HR (95% CI) Age, y <54 5437 1355/42.5 1.0 1124/33.0 0.77 (0.71–0.84) 1082/28.8 0.67 (0.61–0.72) 977/25.6 0.61 (0.56–0.66) 899/23.7 0.56 (0.51–0.62) <0.0001 54–59 7791 1816/56.9 1.0 1597/47.3 0.80 (0.75–0.86) 1480/40.3 0.67 (0.63–0.72) 1493/40.2 0.69 (0.64–0.74) 1405/35.0 0.61 (0.56–0.65) ≥59 10 568 2022/79.6 1.0 2136/72.3 0.85 (0.80–0.90) 2166/67.0 0.78 (0.73–0.83) 2098/64.4 0.75 (0.70–0.80) 2146/61.4 0.72 (0.67–0.76) Cigarettes smoked/d <16 7620 1311/56.8 1.0 1480/48.5 0.79 (0.73–0.85) 1500/43.1 0.70 (0.65–0.76) 1583/40.7 0.66 (0.62–0.71) 1746/37.2 0.60 (0.56–0.65) 0.31 16–20 8057 1699/58.3 1.0 1601/49.6 0.81 (0.75–0.87) 1669/45.1 0.72 (0.67–0.77) 1607/42.8 0.69 (0.64–0.74) 1481/39.5 0.63 (0.58–0.67) >20 8119 2183/58.9 1.0 1776/51.4 0.82 (0.77–0.87) 1559/44.8 0.71 (0.66–0.76) 1378/43.7 0.70 (0.66–0.76) 1223/42.8 0.69 (0.64–0.75) Years smoked, y <33 5938 1283/44.2 1.0 1151/34.9 0.78 (0.72–0.84) 1171/31.1 0.67 (0.62–0.72) 1177/29.1 0.64 (0.59–0.69) 1156/26.6 0.58 (0.53–0.63) 0.0025 33–40 6874 1599/52.4 1.0 1428/45.6 0.84 (0.78–0.90) 1333/39.2 0.72 (0.67–0.78) 1293/38.1 0.70 (0.65–0.76) 1221/35.2 0.63 (0.58–0.68) ≥40 10 984 2311/77.8 1.0 2278/68.8 0.81 (0.76–0.86) 2224/63.4 0.73 (0.69–0.78) 2098/62.5 0.72 (0.68–0.76) 2073/59.3 0.69 (0.64–0.73) Daily alcohol consumption, g <5.3 7225 677/57.5 1.0 1176/53.7 0.94 (0.85–1.03) 1543/47.0 0.80 (0.73–0.88) 1778/44.6 0.77 (0.70–0.84) 2051/40.3 0.71 (0.65–0.78) 0.12 5.3–20.4 7290 1384/58.8 1.0 1534/47.7 0.76 (0.70–0.82) 1496/41.8 0.67 (0.62–0.73) 1493/40.1 0.64 (0.60–0.69) 1383/37.3 0.60 (0.56–0.65) ≥20.4 7524 2647/57.1 1.0 1784/48.3 0.81 (0.76–0.86) 1358/42.6 0.71 (0.66–0.76) 1005/40.3 0.70 (0.65–0.75) 730/37.8 0.64 (0.59–0.70) BMI, kg/m2 <25 9321 1870/63.0 1.0 1614/53.4 0.79 (0.74–0.85) 1795/46.9 0.69 (0.64–0.73) 1893/44.3 0.65 (0.61–0.70) 2149/39.7 0.58 (0.54–0.62) 0.0009 25–28 7415 1461/55.2 1.0 1535/47.5 0.81 (0.75–0.87) 1526/41.5 0.70 (0.65–0.75) 1490/39.8 0.69 (0.64–0.74) 1403/37.6 0.66 (0.61–0.71) ≥28 7042 1858/56.1 1.0 1706/49.0 0.83 (0.78–0.89) 1403/44.4 0.75 (0.70–0.81) 1180/42.5 0.73 (0.68–0.79) 895/41.7 0.70 (0.64–0.76) Trial intervention group Beta carotene 11 867 2620/59.0 1.0 2397/50.7 0.81 (0.76–0.85) 2361/44.5 0.70 (0.66–0.74) 2272/42.4 0.68 (0.64–0.72) 2217/39.2 0.62 (0.59–0.66) 0.51 No beta carotene 11 929 2573/57.4 1.0 2460/49.1 0.81 (0.77–0.86) 2367/44.2 0.73 (0.69–0.77) 2296/42.2 0.70 (0.66–0.74) 2233/39.5 0.65 (0.62–0.69) Alpha-tocopherol 11 955 2590/58.2 1.0 2439/49.8 0.82 (0.77–0.87) 2398/45.0 0.73 (0.69–0.77) 2320/43.5 0.72 (0.68–0.76) 2208/39.4 0.65 (0.61–0.69) 0.34 No alpha-tocopherol 11 841 2603/58.2 1.0 2418/50.0 0.80 (0.76–0.85) 2330/43.7 0.70 (0.66–0.74) 2248/41.2 0.66 (0.62–0.70) 2242/39.4 0.62 (0.59–0.66) Years of follow-up 0–13 9625 2466/40.2 1.0 2036/32.3 0.79 (0.74–0.84) 1856/28.0 0.70 (0.66–0.75) 1691/25.5 0.65 (0.61–0.69) 1576/23.2 0.60 (0.56–0.64) 0.02 13–23 9596 1998/88.6 1.0 1919/71.2 0.80 (0.75–0.85) 1897/61.2 0.69 (0.65–0.74) 1932/60.9 0.71 (0.66–0.75) 1850/54.6 0.63 (0.59–0.68) ≥23 4575 729/135.0 1.0 902/121.2 0.90 (0.82–0.99) 975/104.1 0.77 (0.70–0.85) 945/95.9 0.73 (0.66–0.80) 1024/91.3 0.70 (0.63–0.77)

The results were not materially different when the first 5 years of follow-up were excluded from the analysis (for all-cause mortality in the multivariate model, fifth versus first quintile: HR=0.64; 95% CI, 0.61–0.67; P trend <0.0001; Online Table VIII). Our findings also remained unchanged after excluding from the analysis 12 488 participants who reported a history of CVD, diabetes mellitus, or both (for all-cause mortality in the multivariate model, fifth versus first quintile: HR=0.66; 95% CI, 0.62–0.70; P trend <0.0001; Online Table IX).

Discussion

The primary findings of this large cohort analysis of male smokers were significant inverse associations between prospectively measured serum beta carotene concentrations and overall and cause-specific mortality, with men in the highest serum quintile experiencing 36% lower overall mortality compared with those in the lowest quintile. Of note, the associations were similar for deaths occurring in both the early (0 to <13 years) and later follow-up periods (13–23 and ≥23 years), as well as within most health risk factor subgroups examined, with the exception of somewhat stronger inverse associations in younger men, those with fewer years of smoking, and those with BMI <25 kg/m2 (eg, 44%, 42%, and 42% lower overall mortality for the highest beta carotene quintile, respectively).

To the best of our knowledge, this is the largest investigation of beta carotene biochemical status and mortality. Our findings are consistent with smaller studies of chronic disease mortality. The protective association is similar to a recent meta-analysis of all-cause mortality in 25 468 men and women (6137 deaths), where a relative risk of 0.69 (95% CI, 0.59–0.80) was observed for those in the highest versus lowest serum beta carotene category.5 The NHANES III study (The Third National Health and Nutrition Examination Survey) of 16 008 adults (4225 deaths) found that those in the highest quintile of serum beta carotene experienced a 25% lower risk of death compared with those in the lowest quintile (95% CI, 10%–37%).6 The Swedish Uppsala cohort study of 2301 older men with 630 deaths showed a relative risk of 0.76 for all-cause mortality (95% CI, 0.67–0.85; P=0.0001) per 1-SD increase in serum beta carotene,9 with reduced risk observed for each major cause of death, while the mortality HR for high versus low quartile of beta carotene among 2646 white male asbestos-exposed insulators (984 deaths) was 0.63 (95% CI, 0.51–0.77).7 In addition, a lower HR of 8% per SD increment in plasma beta carotene (95% CI, +1% to −16%; P=0.08) was observed in the British National Diet and Nutrition Survey study of 1054 older men and women (717 deaths).8 Cause-specific mortality findings have not been entirely consistent, with some studies reporting null associations for cancer deaths,7,8 and several relatively small studies showing inverse CVD mortality associations with serum beta carotene,9–13 but not all.6,14 Moreover, there are few reports about respiratory disease,8 diabetes mellitus, and other causes. Given that fruits and vegetables are the primary dietary sources of beta carotene, prior studies that show their higher consumption being associated with reduced risk of overall and cause-specific mortality are supportive of the present biochemical findings.21,22

Large controlled trials have demonstrated that beta carotene supplementation not only had no health benefits, but resulted in unanticipated harmful effects.15–18 The ATBC study, the parent trial of the present cohort serum analysis, and the CARET (Beta-Carotene and Retinol Efficacy Trial) found significantly increased overall mortality (8% and 17%, respectively) among participants who received beta carotene (or beta carotene plus retinyl palmitate in the latter trial) supplements.15,16 By contrast, the Physicians’ Health Study and the Women’s Health Study controlled trials showed no effects of supplemental beta carotene.17,18 The beta carotene dosages administered in these trials had increased the serum concentrations >10× the average,15 raising concerns about natural dietary sources of beta carotene versus supplementation,23 particularly given that at extremely high concentrations beta carotene may lose its antioxidant function and instead have prooxidant effects.24 The aforementioned studies of fruit and vegetable consumption support a mortality benefit for greater dietary sources of beta carotene,21 and the present findings combined with previous observational studies support inverse associations of overall and cause-specific mortality with higher serum beta carotene concentrations within the normal range. For example, the cubic spline regression analysis suggested an optimal relative mortality reduction for older men whose serum beta carotene values were at least 160 µg/L, with a sharper mortality excess for men with values below 160 µg/L. For overall and cause-specific mortality endpoints, our data indicate that decreased mortality is evident with beta carotene concentrations in the 180 to 190 µg/L range and is strongest between 200 and 700 µg/L. Our findings are in line with a previous report of the normal range being 70 to 680 µg/L, with an average concentration of 190 µg/L.25 It should be noted, however, that the male smokers in our study had relatively low beta carotene status (mean and median serum concentrations 212 and 170 µg/L, respectively), potentially providing a better examination of the dose-response relation in the lower range. The relationship between higher beta carotene concentrations and lower mortality risk should be investigated in additional studies that include nonsmokers and women.

Mammals do not synthesize carotenoids de novo, and the main sources of carotenoids (including beta carotene) for humans are of dietary plant origin. For example, most fruits and vegetables contain beta carotene, including carrots, red bell peppers, oranges, sweet potatoes, broccoli, and all green leafy vegetables.26 Among those, carrots may provide an especially important source of dietary beta carotene.26 As a short-term reflection of dietary intake, serum carotenoids are widely accepted as good biomarkers of fruit and vegetable consumption.26,27 Therefore, since supplements were rarely used at baseline, the primary source for beta carotene in this cohort was fruit and vegetable consumption. Fruit and vegetables are the predominant sources of many other bioactive micronutrients, including polyphenols, vitamin C, fiber, folacins, flavonoids, other carotenoids, and several minerals that have multiple biological mechanisms beyond antioxidation, including regulation of detoxification enzymes (CYP monooxygenases), activation of the immune system, modulation of hormone metabolism, and antibacterial and antiviral effects.28 Thus, serum beta carotene may be serving as a biomarker of the beneficial effects of this dietary pattern on health outcomes. Adjustment for fruit and vegetable consumption in our serum beta carotene models did not materially attenuate the estimated mortality associations, however (Online Table I). Likely explanations for this include (1) greater measurement error for self-reported fruit and vegetable intake based on the food frequency questionnaire than for serum beta carotene measured using high-performance liquid chromatography assays; (2) the carotenoid composition of fruits and vegetables and the carotenoid bioavailability based on cooking methods vary, and adjustment for total fruit and vegetable consumption may not have adequately reflected these effects; and (3) host factors that influence serum biochemical status, including gut carotenoid absorption, metabolism, and bioavailability that may confer inter-individual differences.26 On the other hand, it is also biologically plausible for a direct beneficial association for higher beta carotene, which may include antioxidant or anti-inflammatory activity, as oxidative stress and inflammation are involved in diverse pathologies of chronic diseases, including insulin resistance and beta cell dysfunction.29 For example, carotenoids (including beta carotene) inhibit the downstream production of inflammatory cytokines through suppression the NF-κB (nuclear factor-κB) pathway, which can translocate to the nucleus and then activate the inflammation-related genes.26 In addition, beta carotene is a pro-vitamin A precursor of retinoids which exert pleiotropic effects regulating expression of >500 genetic response elements through the retinoic acid and retinoid X nuclear receptors.30

It is noteworthy that the present study showed a stronger serum beta carotene association for CVD mortality than for cancer mortality, in line with some studies of fruit and vegetable consumption.21,31 Beta carotene and other antioxidant compounds are known to inhibit lipid peroxidation in arterial walls, influencing plaque stability, vasomotor function, platelet aggregation, and thrombosis.32,33 In addition, because of its lipophilic and nonpolar properties, beta carotene is exclusively transported by lipoproteins in the bloodstream and is primarily located in lipoprotein cores. Studies have shown that beta carotene is mostly associated with LDLs (low-density lipoproteins),26,34 and oxidative conversion of LDL is considered to have an important role in atherogenesis. Thus, as one of the antioxidants, beta carotene may play a role in the modification of LDL oxidation and lipid peroxidation, consequently lowering CVD risk and mortality.35 Although biologically plausible reasons for the somewhat weaker cancer mortality association are not clear, it is possible that serum beta carotene status is overshadowed by other risk factors, such as cigarette smoking, something supported by our observation of a stronger inverse cancer mortality association among men with fewer cumulative years of smoking. Examination of specific cancer sites could shed light on this; that is, is the serum beta carotene association weaker in smoking-related malignancies, such as lung, oropharynx, larynx, and bladder. Our results are consistent with previous studies, demonstrating inverse or positive associations of beta carotene with diabetes mellitus36 and lung function,37 respectively, and higher fruit and vegetable consumption have been associated with risk of chronic obstructive pulmonary disease among current and ex-smokers.38 Of note, we observed a strong inverse association between serum beta carotene and diabetes mellitus mortality, despite the small number of diabetes deaths (ie, 119), consistent with a meta-analysis showing greater green leafy vegetable consumption associated with lower type 2 diabetes mellitus mortality,22 possibly through reduced systemic oxidative stress. How higher serum beta carotene might be related to lower risk of deaths from injuries and accidents could involve improved survival after the initial events, either directly (eg, anti-inflammatory effects) or indirectly through better general health. However, we could not preclude the possibility that the association may be a chance finding or owing to residual confounding, although previous cross-sectional studies reported that a history of attempted suicide was associated with lower serum carotenoid levels.39

There are several notable strengths of our investigation. The large sample size and complete long-term ascertainment of cause-specific deaths through national registries provided validity and substantial power for detecting moderate serum beta carotene associations with overall and cause-specific mortality and for evaluating effect modification by other risk factors. High-quality isocratic high-performance liquid chromatography biochemical assays conducted early in the study provided serum beta carotene concentrations for >29 000 men in our cohort that were more accurate than self-reported dietary food frequency questionnaire data. Study limitations include the homogenous smoker population of Finnish males which reduces generalizability of our findings to other populations, and the use of a single baseline serum beta carotene measurement, with possible changes in biochemical status and diet during follow-up. This nondifferential misclassification would, however, bias the findings toward the null. The correlation between serum concentrations at baseline and 3 years after randomization among those who were not supplemented with beta carotene was high, however (Spearman correlation coefficient=0.70, P<10–5), supporting its relative stability over time. Finally, although we carefully controlled for measured covariates and potential confounding factors, we still cannot rule out the possibility of residual confounding, including by unmeasured factors, that may have biased the observed associations.

In conclusion, we found significant inverse associations between serum beta carotene concentrations and all-cause and cause-specific mortality, including death from CVD, heart disease, stroke, and cancer. The dose-response associations over a 30-year period were not attenuated by adjustment for important risk factors. Our results provide evidence to support an association between higher beta carotene serological status within the normal range and reduced mortality. Whether low-dose beta carotene supplementation could be similarly associated with lower mortality would require evidence from controlled trials. The association should be reexamined in other study populations that include women, nonsmokers, and other ethnic/racial participants.

Nonstandard Abbreviations and Acronyms ATBC Alpha-Tocopherol, Beta-Carotene Cancer Prevention BMI body mass index CARET Beta-Carotene and Retinol Efficacy Trial CVD cardiovascular disease HDL high-density lipoprotein HR hazard ratios ICD International Classification of Diseases LDL low-density lipoproteins NF-κB nuclear factor-κB

Acknowledgments

We appreciate all participants in the Alpha-Tocopherol, Beta-Carotene Cancer Prevention cohort for their contribution to the study.

Sources of Funding The Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study is supported by the Intramural Research Program of the US National Cancer Institute, National Institutes of Health, and by US Public Health Service contract HHSN261201500005C from the National Cancer Institute, Department of Health and Human Services.

Disclosures None.

Footnotes