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. Author manuscript; available in PMC: 2015 Feb 1.
Published in final edited form as: J Bone Miner Res. 2014 Feb;29(2):408–417. doi: 10.1002/jbmr.2041

Protective effects of dietary carotenoids on risk of hip fracture in men: the Singapore Chinese Health Study

Zhaoli Dai 1, Renwei Wang 2, Li-Wei Ang 3, Yen-Ling Low 1, Jian-Min Yuan 2,4, Woon-Puay Koh 1,5
PMCID: PMC3894263  NIHMSID: NIHMS508906  PMID: 23857780

Abstract

Experimental and epidemiologic data suggest that carotenoids in vegetables and fruits may benefit bone health due to their antioxidant properties. The relationship between dietary total and specific carotenoids, as well as vegetables and fruits, and risk of hip fracture was examined among Chinese in Singapore. We used data from the Singapore Chinese Health Study, a prospective cohort of 63,257 men and women who were of ages 45–74 years between 1993 and 1998. At recruitment, subjects were interviewed on lifestyle factors and medical history. Usual diet was measured using a validated food frequency questionnaire. During a mean follow-up of 9.9 years, we identified 1,630 hip fracture incident cases. Among men, consumption of vegetables was associated with lower hip fracture risk. Similarly, dietary total carotenoids and specific carotenoids, α-, β-carotene and lutein/zeaxanthin were inversely associated with hip fracture risk. Compared to men in the lowest quartile of nutrient density, men in the highest quartile had statistically significant 26% to 39% risk reduction (all Ps for trend < 0.05). When stratified by body mass index, the greatest protective effects of total vegetables and carotenoids were found in men with body mass index < 20kg/m2 (P for trend≤0.004). There was no association between dietary carotenoids or vegetables/fruits and hip fracture risk among women. This study suggests that adequate intake of vegetables may reduce risk of osteoporotic fractures among elderly men and that the antioxidant effects of carotenoids may counteract the mechanism of osteoporosis related to leanness.

Keywords: carotenoids, vegetables, hip fracture, body mass index, Chinese

Introduction

Osteoporosis is an epidemic problem affecting both elderly men and women worldwide. Although the prevalence of osteoporosis and related fractures is higher in women than in men, men experience greater comorbidity and mortality after hip fracture than women (1,2). Studies have shown that osteoporosis in men is under-recognized and under-studied epidemiologically and clinically (3–5). Hence, understanding the risk factors and the underlying mechanism of hip fracture would help to develop a preventive strategy for osteoporotic fractures.

Evidence from in vitro and in vivo studies suggests that reactive oxygen species (ROS) may increase osteoclastogenesis (6,7) and osteoclastic differentiation (7,8), or suppress osteoblastic differentiation (9,10). Therefore, increased oxidative stress may play an important role in age-related bone loss (11,12) that leads to osteoporotic fractures. Dietary antioxidant carotenoids, primarily derived from fruits and fresh vegetables, have been reported to promote bone formation in experimental studies (13,14), and to protect against osteoporosis and related fractures in some but not all epidemiological studies (15–20). Additionally, lycopene, a carotenoid, was shown to inhibit osteoclast formation in rat bone marrow cultures (21), and was negatively related to bone resorption biomarkers and oxidative stress in postmenopausal women (22,23). Taken together, these data raised a hypothesis that antioxidant carotenoids may counteract the adverse effect of ROS in bone turnover, and therefore can potentially protect against osteoporotic fractures.

Leanness has been established as an independent risk factor of hip fracture (24–26). Recently, several observational studies have reported that low body mass index (BMI) could be related to increased oxidative stress (27–29), particularly, in men (28). Hence, in the present study, we aimed to assess the association between dietary total and specific carotenoids, including α- and β-carotene, β-cryptoxanthin, lycopene and lutein/zeaxanthin, and risk of hip fracture among elderly men and women. In addition, we also evaluated whether BMI modified the effects of these dietary components on the risk of hip fracture among elderly of an Asian population, who generally have lower BMI than their Western counterparts (30).

Methods

Study population

This study was conducted in the Singapore Chinese Health Study, a community-based prospective cohort study to investigate diet, lifestyle factors and risk of chronic diseases. We enrolled 63,257 middle-aged to older men (n=27,959) and women (n=35,298) between April 1993 and December 1998. The study participants were restricted to two major dialect groups in Singapore, which are the Hokkiens and the Cantonese who originated from Fujian and Guangdong provinces in Southern China. During the enrollment period, all of our study participants were residents of government housing estates, where 86% of the Singapore population was housed. This study was approved by the Institutional Review Board at the National University of Singapore, and all enrolled subjects gave informed consent.

Baseline assessment was conducted through a face-to-face structured interview during the initial enrollment. Information was recorded by a trained interviewer using a structured questionnaire that included demographics, medical history, cigarette smoking, alcohol consumption, physical activity, and detailed menstrual and reproductive history (women only). Body weight and height at baseline were self-reported during the interview. There were 9,781 cohort participants with unknown weight, 97 with unknown height, and 192 with both unknown weight and height. BMI is calculated as weight in kilograms divided by height in meters squared. For those with missing weight and/or height, BMI was calculated using imputed weight and/or height derived from the linear regression equation: Weight = y-intercept + gradient × height, where values for the y-intercept and gradient were derived from gender-specific weight-height regression lines obtained from all cohort participants with known heights and weights. This method of imputed BMI was reported in detail previously (31).

Dietary intake was recorded using a 165-item semi-quantitative Food Frequency Questionnaire (FFQ), which incorporates common and distinct food items in Singapore. The FFQ had been validated previously using 24-hour recalls and re-administration of the FFQ among a subset of 810 participants (32). The validation study by these two methods showed similar distributions with most mean pairs for energy and nutrients within 10% of each other's values. The correlation coefficient by these two methods for each dietary component ranged between 0.24 and 0.79, which is comparable with previous validation study in diverse populations (33). The dietary intake of each nutrient was derived based on the Singapore Food Composition Database, which has been described in detail previously (32). This database was developed specifically for this cohort study and listed approximately 100 nutritional and non-nutritional values per 100 g of the edible raw and cooked foods. The physical activity portion of the questionnaire was modeled after the European Prospective Investigation in Cancer (EPIC) study physical activity questionnaire, which has been shown to be valid and reproducible (34). More information about questionnaire used in this cohort can be found in supplemental material online.

Case ascertainment

Hip fracture cases were identified via record linkage analysis with hospital discharge databases of the MediClaim System, which captures inpatient discharge information from all public and private hospitals in Singapore. All cases were verified manually by surgical records or medical records. As of December 31, 2010, only 47 subjects from this cohort were known to be lost to follow-up due to migration out of Singapore or for other reasons. After excluding four cases of traumatic fractures from road traffic accidents, and one case due to cancer metastasis in the femur, 1,733 hip fracture cases were identified through record linkage. We excluded from statistical analysis of 103 prevalent cases of hip fracture, which occurred before enrollment to the cohort for these participants. Thus, 1,630 fracture cases and 61,524 subjects without fractures were included in the final analysis. The causes and date of death of all cohort participants were ascertained through record linkage with the population-based Singapore Registry of Births and Deaths.

Statistical analysis

For each study subject, person-years were counted from the date of baseline interview to the date of hip fracture diagnosis, death, migration or 31 December 2010, whichever occurred first. The Cox proportional hazards regression was used to assess the association between baseline dietary carotenoids, fruits and vegetables, and hip fracture risk by comparing higher quartiles to the lowest quartile (35). Quartiles of each energy-adjusted food or nutrient were based on the distribution of both men and women combined in the whole cohort. The strength of the association between a dietary factor and hip fracture risk was estimated by hazard ratios (HRs) and their corresponding 95% confidence intervals (CI). To examine linear trend, ordinal values of the quartile of each dietary component was entered as a continuous variable in the Cox proportional hazards model. We did not identify any violation of the proportional hazard assumption or multicollinearity among the covariates that were entered in the models.

All models in the analyses included the following covariates: age (continuous), year at recruitment (1993–1995 and 1996–1998), dialect group (Hokkien, Cantonese), level of education (no formal education, primary school, secondary school or higher), BMI (kg/m2, continuous), smoking status (never smokers, ex-smokers, current smokers), moderate physical activity (none, 2–3 hrs weekly, 4+ hrs weekly), calcium (mg/1,000 kcal/day), soy isoflavones (mg/1,000 kcal/day), pyridoxine ((mg/1,000 kcal/day), total energy intake (kcal/day), menopause status (women only; yes, no), use of hormone replacement therapy at recruitment (women only; yes, no), baseline self-reported physician-diagnosed history of diabetes mellitus (yes, no) and stroke (yes, no). Dialect group was a recruitment criterion. In addition, Cantonese men appeared to have lower hip fracture risk than the Hokkien men (p<0.01), although there was no difference in hip fracture incidence in women regardless of dialect groups (p=0.39). Hence, dialect group was also included as a covariate in our models. Adjustment for total energy intake in addition to the energy-adjusted dietary component (exposure of interest) is based on the multivariate nutrient density model (35). We used the multivariate nutrient density model proposed by Willet et al (35) to assess the diet-hip fracture associations in this study. The use of nutrient density, in which nutrient intake is divided by total energy intake, is an accepted measure for studying nutrient intake, and accounts for total energy intake in nutritional studies and epidemiologic analyses. As explained by Willet et al. (35), the nutrient density method has several advantages: it can be calculated directly for an individual without the use of any statistical models; it is familiar to nutritionists as a measure of dietary composition; and it has been used in national dietary guidelines.

To study the BMI-hip fracture risk association and to assess the modifying effects of BMI on the diet-hip fracture risk associations, we only included participants with known body weight and height in these analyses. Hence, 52,792 cohort participants who reported body weight and height at baseline were included in the analysis including 1,238 hip fracture cases in this subgroup. Subjects were stratified by BMI levels of < 20, 20 – < 25, and ≥ 25 kg/m2, which is based on the previous finding of BMI < 20 kg/m2 being a risk factor for hip fracture from this cohort (26) and a meta-analysis of 12 population-based cohort studies (24); and that BMI ≥25 kg/m2 being the current cut-off for overweight proposed by the World Health Organization (36) which is applicable to Asian populations (30). Heterogeneity of the diet-hip fracture risk associations among different BMI levels was tested using an interaction term (product between diet and BMI) included in the Cox model. Finally, we performed a sensitivity analysis for the diet-hip fracture risk association after excluding subjects who had reported extreme energy intakes (≤ 600 and ≥ 3000kcals). All statistical analysis was conducted using SAS Version 9.2 (SAS Institute, Inc., Cary, North Carolina). All reported p values are two-sided. For the present analysis, we had a priori hypothesis that total and specific carotenoids were protective against hip fracture risk. The p values were thus not adjusted for multiple testing; p < 0.05 was considered statistically significant.

Results

Among the 1,630 incident hip fracture cases, the mean time interval between cohort enrollment and hip fracture diagnosis was 9.9 [standard deviation (SD) 4.5] years. The mean age at fracture was 74.4 (SD 7.5) years. Women accounted for 72.4% of all hip fractures and the age-standardized incidence rates of hip fractures in women (234 per 100,000 person-years) was twice that in men (123 per 100,000 person-years). In the whole cohort, compared to women, men were older in age, more educated, and had greater proportion of habitual smokers and alcohol drinkers. The mean BMI in the study population was 23 kg/m2. BMI levels were similar between men and women, as were physical activity and prevalence of diabetes mellitus and stroke (Table 1). Seventy-two percent of women were post-menopausal at enrollment, but only 3.7% of them reported use of hormone replacement therapy (Table 1). Regarding dietary intakes, according to the US RDA values (37), both men and women in our cohort had adequate intake of protein. Intakes of vegetables were comparable between the two genders, although fruit intake was higher in men than in women. Overall, absolute dietary intake of total carotenoids for men was about 5% higher than women. Among all carotenoids that were examined, β-carotene had the highest correlation with total carotenoids, followed by lutein/zeaxanthin and lycopene. Among specific carotenoids, β-carotene was highly correlated with α-carotene and lutein. Total carotenoids were better correlated with vegetables than with fruits. The correlation between different types of vegetables and carotenoids reflect the dietary source of the carotenoids; green vegetables were best correlated with lutein/zeaxanthin, and yellow-orange vegetables with α-carotene (see Pearson's correlation coefficients between dietary intake of carotenoids and food groups/specific vegetable or fruit item in Supplemental Table 1).

Table 1.

Baseline characteristics of men and women who participated in the Singapore Chinese Health Study (n=63,154)

Characteristics Men (n=27,913) Women (n=35,241)
Age at recruitment, years, mean (SD) 59.6 (7.9) 56.3 (8.0)
Body mass index, mean (SD) 23.0 (3.2) 23.2 (3.3)
 <20 kg/m2(%) 16.1 14.9
 20–25 kg/m2(%) 62.3 63.2
 ≥25 kg/m2(%) 21.6 21.9
Level of education (%)
 Secondary school or higher 37.9 20.7
Smoking status (%)
 Ever smokers 58.1 8.8
Alcohol consumption (%)
 Daily drinkers 6.4 1.2
Physical activity (%)
 No 75.3 79.9
 0.5–3hr per week 15.4 12.7
 ≥4 hr per week 9.3 7.4
Diabetes mellitus (%) 8.7 9.2
Stroke (%) 1.8 1.2
Postmenopausal (%) 72.4
Hormone replacement use, yes (%) 3.7
Dietary intake
 Total energy intake (kcal), mean(SD) 1749.7 (608.9) 1399.3 (472.0)
 Total protein intake (g), mean (SD) 65.2 (25.9) 54.4 (21.5)
 Total vegetables (g/day), mean (SD) 111.3 (65.6) 110.0 (61.9)
  Green vegetables (g/day), mean (SD) 67.4 (41.6) 66.3 (39.6)
  Yellow-orange vegetables (g/day), mean (SD) 7.3 (8.4) 8.5 (8.8)
  Cruciferous vegetables(g/day), mean (SD) 44.1 (29.6) 43.4 (28.3)
  Tomato products(g/day), mean (SD) 7.7 (9.5) 7.3 (9.1)
 Total fruit (g/day), mean (SD) 212.7 (177.1) 194.4 (162.2)
 Total carotenoids (μg/day), mean (SD) 5800.4 (3884.0) 5541.6 (3495.8)
  α-carotene (μg/day), mean (SD) 240.9 (275.8) 268.2 (287.9)
  β-carotene (μg/day), mean (SD) 2125.6 (1514.3) 2197.4 (1472.7)
  β-crytoxanthin(μg/day), mean (SD) 270.3 (343.0) 240.8 (327.6)
  Lycopene (μg/day), mean (SD) 1307.0 (1707.6) 950.3 (1300.2)
  Lutein/Zeaxanthin (μg/day), mean (SD) 1856.6 (1143.0) 1885.0 (1123.7)
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Table 2 shows the relations between dietary vegetables, fruits and carotenoids, and hip fracture risk for men and women separately. There was a statistically significant, dose-dependent inverse relationship with hip fracture risk for total, green or yellow-orange vegetables in men (p for trend≤0.002), but not in women. Men in the highest quartile of total vegetables had approximately 40% lower in hip fracture risk than men in the lowest quartile. Consumption of tomato products or fruits was not associated with hip fracture risk in either gender. Similarly, statistically significant inverse associations between nutrient densities of total or specific carotenoids, including α-, β-carotene and lutein/zeaxanthin, and risk of hip fracture were seen in men (p for trend < 0.05), but not in women. The strongest association was observed in dietary β-carotene. Compared with men in the lowest quartile, men in the 3rd or 4th quartile of β-carotene experienced a statistically significant 37% decrease in risk of hip fracture (p ≤ 0.005). Nutrient density of β-cryptoxanthin or lycopene was not associated with hip fracture risk in either men or women. The results were essentially the same using gender-specific cut-points for the definition of quartiles for the food and nutrient variables included in this study (data not shown). Expectantly, the consumption of total vegetables and carotenoids were highly correlated in our cohort with a Pearson's correlation coefficient of 0.84. We hypothesized that the protective effects of vegetables were mediated via their carotenoids. When we put both intake levels of vegetables and specific carotenoid, such as β-carotene, in the same model, there was a loss of statistical significance due to the high collinearity of both variables. However, the association remained stronger for β-carotene (p for trend=0.081) than for vegetables (p for trend=0.27), supporting our hypothesis that the protective effect of vegetables was likely to be mediated by carotenoids such as β–carotene. Finally, we re-analyzed the data by treating food/nutrient densities as continuous variables, and the results were essentially the same. The p values for the association between dietary carotenoid and hip fracture risk were 0.0055 for men and 0.35 for women.

Table 2.

Dietary intake of vegetables, fruits and carotenoids in relation to hip fracture incidence for men (n=27,913) and women (n=35,241), The Singapore Chinese Health Study, 1993–2010.

Men (quartile of energy-adjusted intake) Women (quartile of energy-adjusted intake)
Q1 Q2 Q3 Q4 p-trend Q1 Q2 Q3 Q4 p-trend
Total vegetables
Cases 186 121 89 54 285 294 326 275
HR 1.00 0.87 0.74 0.59 0.0006 1.00 0.94 1.04 0.89 0.424
(95% CI) (0.69–1.10) (0.56–0.96) (0.42–0.81) (0.80–1.11) (0.88–1.23) (0.74–1.07)
Green vegetables
Cases 180 121 89 60 286 289 302 303
HR 1.00 0.88 0.76 0.64 0.002 1.00 0.98 1.04 1.02 0.712
(95% CI) (0.70–1.12) (0.58–0.99) (0.47–0.88) (0.83–1.16) (0.88–1.23) (0.85–1.21)
Yellow-orange vegetables
Cases 170 129 99 52 281 308 300 291
HR 1.00 0.93 0.85 0.56 0.0008 1.00 0.96 0.88 0.90 0.143
(95% CI) (0.74–1.17) (0.66–1.09) (0.41–0.77) (0.82–1.13) (0.74–1.03) (0.76–1.07)
Tomato products
Cases 131 131 110 78 345 291 296 248
HR 1.00 1.03 1.01 0.84 0.304 1.00 1.00 1.01 0.90 0.272
(95% CI) (0.80–1.31) (0.78–1.31) (0.63–1.12) (0.85–1.17) (0.86–1.18) (0.76–1.06)
Total fruits
Cases 130 129 98 93 389 282 251 258
HR 1.00 1.10 0.91 0.96 0.533 1.00 0.94 0.93 0.97 0.677
(95% CI) (0.85–1.41) (0.69–1.22) (0.70–1.31) (0.80–1.10) (0.79–1.11) (0.81–1.17)
Total carotenoids
Cases 177 122 93 58 329 310 288 253
HR 1.00 0.85 0.80 0.63 0.006 1.00 0.97 0.98 0.95 0.642
(95% CI) (0.67–1.08) (0.61–1.04) (0.45–0.88) (0.83–1.14) (0.83–1.16) (0.79–1.15)
α-Carotene
Cases 166 117 107 60 273 303 309 295
HR 1.00 0.83 0.88 0.61 0.005 1.00 0.91 0.97 0.97 0.927
(95% CI) (0.65–1.05) (0.68–1.12) (0.45–0.83) (0.77–1.07) (0.82–1.15) (0.81–1.15)
β-Carotene
Cases 183 134 76 57 282 319 291 288
HR 1.00 0.96 0.63 0.63 0.0003 1.01 0.96 0.97 0.616
(95% CI) (0.76–1.20) (0.48–0.83) (0.46–0.87) 1.00 (0.86–1.19) (0.81–1.14) (0.81–1.17)
β-Cryptoxanthin
Cases 114 123 110 103 376 266 231 307
HR 1.00 1.12 1.07 0.95 0.716 1.00 0.99 0.96 1.18 0.103
(95% CI) (0.86–1.45) (0.81–1.41) (0.71–1.27) (0.84–1.16) (0.80–1.14) (1.00–1.39)
Lutein/Zeaxanthin
Cases 166 132 92 60 277 285 286 332
HR 1.00 1.04 0.87 0.74 0.049 1.00 0.95 0.95 1.03 0.673
(95% CI) (0.82–1.32) (0.67–1.13) (0.54–1.02) (0.80–1.12) (0.80–1.13) (0.87–1.23)
Lycopene
Cases 124 129 116 61 457 341 212 170
HR 1.00 1.15 1.04 0.91 0.538 1.00 1.08 0.87 0.88 0.07
(95% CI) (0.89–1.47) (0.80–1.35) (0.68–1.24) (0.94–1.25) (0.73–1.02) (0.73–1.07)
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Hazard ratios (HRs) were adjusted for age at recruitment (years), year of recruitment (1993–1995, 1995–1998), dialect group (Hokkien, Cantonese), body mass index (kg/m2), level of education in categories (no formal education, primary school, secondary school or higher), total energy intake (kcal), smoking status (never smokers, ex-smokers, current smokers), physical activity (none, 2–3 hrs weekly, ≥4hrs weekly), calcium (quartiles, mg/1,000kcal/day), soy isoflavones (quartiles, mg/1,000kcal/day), vitamin B6 (quartiles, mg/1,000kcal/day), menopausal status (women only; yes, no), use of hormone replacement therapy at recruitment (women only; yes, no), baseline physician-diagnosed history of diabetes (yes, no) and stroke (yes, no). CI, confidence interval.

BMI was inversely related to risk of hip fracture in men (Table 3). Compared with BMI < 20kg/m2, men with BMI 20 – < 25 and ≥ 25 kg/m2 had hip fracture risk reduced by 18% and 33%, respectively (p for trend = 0.016). There was a 9% reduction of hip fracture risk for women with BMI ≥ 25 kg/m2 compared with women with BMI < 20 kg/m2, but the difference was not statistically significant between two genders (p for interaction=0.137) (Table 3). When BMI was analyzed on a continuous scale, for each unit increase in BMI, risk of hip fracture was reduced by 5.5% [95% CI=0.92–0.98] in men, and only by 1.1% [95% CI=0.97–1.01] in women. Further adjustment for dietary protein did not materially change the effect size of this BMI-hip fracture association.

Table 3.

Body mass index in relation to hip fracture incidence in men and women, The Singapore Chinese Health Study, 1993–2010.

Participants with reported BMI only (n=52,792)
Men(n=23,918) Women (n=28,874)
BMI (kg/m2) Cases HR 95% CI Cases HR 95% CI
<20 85 1.00 164 1.00
20–<25 211 0.82 0.64–1.06 468 0.93 0.78–1.11
≥25 70 0.67 0.49–0.93 240 0.91 0.74–1.11
p for trend 0.016 0.384
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Hazard ratios (HRs) were adjusted for age at recruitment (years), year of recruitment (1993–1995, 1995–1998), dialect group (Hokkien, Cantonese), level of education in categories (no formal education, primary, secondary or higher), smoking status (Never smokers, Ex-smokers, Current smokers), total energy intake (kcal/day), physical activity (none, 2–3 hrs weekly, ≥4hrs weekly), calcium (quartiles, mg/1,000kcal/day), soy isoflavones (quartiles, mg/1,000kcal/day), vitamin B6 (quartiles, mg/1,000kcal/day), baseline physician-diagnosed history of diabetes (yes, no) and stroke (yes, no). HR*, adjusted for above plus menopausal status (women only; yes, no) and use of hormone replacement therapy at recruitment (women only; yes, no). CI, confidence interval.

Figure 1 shows the associations between total vegetables/carotenoids and hip fracture risk stratified by BMI for men. Among men with BMI < 20 kg/m2, HRs (95% CIs) of hip fracture for the 2nd, 3rd, and 4th quartile of total vegetable consumption were 0.60 (0.35–1.03), 0.49 (0.26–0.92), and 0.22 (0.08–0.64), respectively, compared with the 1st quartile (p for trend = 0.0008). A null association was observed between total vegetables and hip fracture risk in men with BMI ≥ 25 kg/m2. The difference in the protective effects of total vegetables by BMI categories was statistically significant (p for interaction=0.011). Similarly, the associations between total carotenoids and reduced hip fracture risk in men by BMI categories were also statistically different (p for interaction=0.022). Using total vegetables/carotenoids as a continuous variable for the diet-hip fracture association in each BMI stratum yielded essentially the same results, p=0.0002 and p=0.0086 for total vegetables and total carotenoids, respectively, for men with BMI <20 kg/m2.

Figure 1.

Figure 1

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Hazard ratio (95% CI) for hip fracture by quartiles (Q1,Inline graphic; Q2,Inline graphic; Q3,Inline graphic; Q4,Inline graphic) of dietary intake of total vegetables (Panel A) or total carotenoids (Banel B) in men (n= 23,918), according to levels of body mass index.

(A) Total vegetables in relation to hip fracture risk. Test for trend, p=0.004, 0.87, and 0.68 for BMI<20, BMI=20–<25, and BMI≥25 kg/m2, respectively. (B) Total carotenoids in relation to hip fracture risk. Test for trend, p=0.0008, 0.22, and 0.82 for BMI<20, BMI=20–<25, and BMI≥25 kg/m2, respectively. Model was adjusted for age at recruitment, year of recruitment, dialect group, level of education in categories, smoking status, physical activity, total energy intake, calcium, soy isoflavones, vitamin B6, baseline physician-diagnosed history of diabetes and stroke.

For vegetable subtypes, the inverse association between dietary green or yellow-orange vegetables and hip fracture risk was present in men with BMI < 20 kg/m2, but generally attenuated in men of the higher BMI categories. For specific carotenoids, α-, β-carotene or lutein/zeaxanthin, the reduction in hip fracture risk was again the greatest in men with BMI <20 kg/m2 (Table 4). On the other hand, the associations between fruits, vegetables and carotenoids (total and specific) and hip fracture risk were generally null across all strata of BMI among women (Supplemental Table 2). Further adjustment for dietary protein did not materially alter any results described (data not shown). In addition, we also conducted sensitivity analyses excluding those with extreme caloric intake, and the findings were materially unchanged (data not shown).

Table 4.

Dietary intake of vegetables and carotenoids in relation to hip fracture incidence for men (n=23,918), according to levels of body mass index, The Singapore Chinese Health Study, 1993–2010.

BMI<20 kg/m2 20≤BMI<25 kg/m2 BMI≥25 kg/m2
Cases (n=85) HR 95% CI Cases (n=211) HR 95% CI Cases (n=70) HR 95% CI
Green vegetables (g/1000kcal/day)
Q1 (<28) 45 1.00 76 1.00 16 1.00
Q2 (28–40) 27 0.97 0.59–1.59 51 0.90 0.62–1.29 25 1.72 0.91–3.26
Q3(40–55) 9 0.35 0.17–0.75 46 0.93 0.64–1.36 17 1.43 0.70–2.90
Q4 (≥55) 4 0.23 0.08–0.66 38 0.91 0.60–1.39 12 1.29 0.58–2.86
p for trend 0.0004 0.67 0.604
P for interaction 0.004
Yellow-orange vegetables (g/1000kcal/day)
Q1(<1.8) 42 1.00 65 1.00 28 1.00
Q2 (1.8–3.7) 23 0.77 0.46–1.30 65 1.14 0.81–1.62 19 0.76 0.42–1.36
Q3 (3.7–6.8) 11 0.44 0.22–0.86 50 1.06 0.72–1.54 18 0.86 0.47–1.57
Q4 (≥6.8) 9 0.49 0.23–1.04 31 0.81 0.52–1.25 5 0.27 0.10–0.70
p for trend 0.009 0.42 0.02
P for interaction 0.752
α- carotene (μg/1000kcal/day)
Q1 (<59.8) 41 1.00 65 1.00 23 1.00
Q2 (59.8–114.4) 20 0.66 0.39–1.14 57 0.96 0.67–1.34 21 0.96 0.52–1.74
Q3 (114.4–212.3) 16 0.65 0.36–1.18 53 1.04 0.72–1.51 16 0.81 0.42–1.57
Q4 (≥212.3) 8 0.41 0.19–0.89 36 0.87 0.57–1.34 10 0.60 0.28–1.29
p for trend 0.02 0.70 0.18
P for interaction 0.406
β- carotene (μg/1000kcal/day)
Q1 (<850.4) 48 1.00 69 1.00 24 1.00
Q2 (850.4–1235.4) 21 0.69 0.41–1.18 66 1.20 0.85–1.70 20 0.94 0.51–1.73
Q3 (1235.4–1772.4) 11 0.42 0.21–0.84 39 0.82 0.54–1.25 16 0.92 0.47–1.80
Q4 (≥1772.4) 5 0.27 0.10–0.70 37 1.03 0.66–1.61 10 0.68 0.31–1.53
p for trend 0.0008 0.68 0.41
P for interaction 0.054
Lutein/Zeaxanthin (μg/1000kcal/day)
Q1 (<781.8) 41 1.00 67 1.00 18 1.00
Q2 (781.8–1104.2) 28 1.07 0.65–1.75 62 1.22 0.85–1.74 22 1.48 0.78–2.80
Q3 (1104.2–1544.1) 10 0.46 0.22–0.93 44 1.00 0.68–1.49 17 1.32 0.66–2.63
Q4 (<1544.1) 6 0.40 0.16–0.97 38 1.10 0.71–1.70 13 1.38 0.64–3.01
p for trend 0.009 0.87 0.45
P for interaction 0.015
Lycopene (μg/1000kcal/day)
Q1 (<191.9) 24 1.00 59 1.00 11 1.00
Q2 (191.9–451.9) 29 1.53 0.87–2.67 52 0.97 0.66–1.41 23 1.81 0.87–3.75
Q3 (451.9–858.4) 17 0.94 0.50–1.77 62 1.11 0.77–1.61 21 1.52 0.72–3.23
Q4 (≥858.4) 15 1.28 0.65–2.56 38 0.82 0.53–1.28 15 1.26 0.54–2.91
p for trend 0.85 0.64 0.80
P for interaction 0.844
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Hazard ratios (HRs) were adjusted for age at recruitment, year of recruitment, dialect group, level of education, smoking status, physical activity, total energy intake, calcium, soy isoflavones, vitamin B6, baseline physician-diagnosed history of diabetes and stroke. CI, confidence interval.

Discussion

The present study demonstrate that there was a statistically significant inverse relationship for hip fracture risk in men with dietary total vegetables, green vegetables and yellow-orange vegetables, as well as total and specific carotenoids, α-, β-carotene and lutein/zeaxanthin. In addition, BMI modified the protective effect of total vegetables/carotenoids against hip fracture risk, with an apparent protective effect of vegetables/carotenoids on hip fracture risk for lean men (<20 kg/m2), but not for their heavier counterparts.

This study population had comparable vegetable intake with other Western populations (17,38), although intake of carotenoids was lower (16). This is possibly due to relatively low prevalence of supplement use in this cohort (only about 5% of men and 8% of women reported use of oral supplements). More than three quarters of total carotenoids in this population were sourced from dietary intake of total vegetables, and another 10% from fruit. Green vegetables provided 62% and 80% of β-carotene and lutein/zeaxanthin, respectively; 72% of α-carotene was provided by yellow-orange vegetables; fruits contributed 99% of β-cryptoxanthin; melons and tomato products contributed 47% and 29% of lycopene, respectively.

Although several cross-sectional studies reported a positive association between vegetables and reduced hip fracture risk (39–41), most of the results in prospective cohort or intervention studies failed to reach statistical significance (38,42,43). A recent systematic review also concluded no clear beneficial effects of fruit and vegetables on bone health in postmenopausal women over 45 years of age (44). This concurs with our null findings among women, where the protective effects of carotenoids or vegetables were weakened and became non-statistically significant after further adjustment for other lifestyle and dietary covariates. This suggests that other lifestyle factors, particularly other dietary nutrients, may confound the association between carotenoids/vegetables and hip fracture risk for women. We have previously reported dietary isoflavones (26) and pyridoxine (45) as significant protective factors for hip fracture among women only. We postulate the plausible mechanisms for these two nutrients are related to their effects on estrogen, which play a critical and dominant role in bone turnover in women (46). Thereby, it is possible that the effect of oxidative stress on bone health may be less pronounced in postmenopausal women, which may explain the null association between carotenoids/vegetables and hip fracture risk for women. In the Framingham Osteoporosis Study, statistically significant protective effect of total carotenoids on hip fracture risk was reported when men and women were combined in the analysis (16). Nevertheless, our finding of an inverse association between vegetable/carotenoid and hip fracture risk in men is generally consistent with other findings from the Framingham study that also supported a stronger protective role of carotenoids in men. In the Framingham Study, dietary vegetables reduced longitudinal bone loss at the trochanter and Ward's area in men, but not in women (41). The Framingham study also reported that more apparent protective effects from multiple carotenoids, including total carotenoids, β-carotene, lycopene and lutein/zeaxanthin, were significantly related to trochanter BMD in men but less so in women (17). Furthermore, testosterone may increase the bioavailability of carotenoids (47). Our observed protective effect of carotenoids on hip fracture risk in men only could reflect the male hormonal effect on carotenoid uptake and biological effect.

We propose that the benefits of carotenoids on bone might be via their counteractive effects against oxidative stress, which has been shown to play a possible etiologic role in age-related bone loss and consequent osteoporotic fractures (12) by increasing osteoclastogenesis and stimulating osteoclastic differentiation (7,8) through the nuclear factor-kappaB (NF-κB) ligand (RANKL) expression and signaling mechanism. Besides the antioxidant function of carotenoids, other mechanisms of non-antioxidant functions can also be responsible for their roles in bone health. In vitro and animal studies have demonstrated that the carotenoid family may exert dual anti-catabolic and pro-anabolic activities in bone by antagonizing the same NF-κB signal transduction pathway to suppress osteoclast differentiation and promote osteoblast mineralization (48). Several in vitro studies have also reported that β-carotene, lycopene and β-cryptoxanthin may have direct stimulatory effects in the proliferation, differentiation and alkaline phosphatase activity of osteoblasts (13,49,50).

In addition to the high carotenoid content in vegetables, another possible mechanism for the beneficial effects on bones includes the alkaline nature of vegetables. In particular, potassium in vegetables may neutralize excess metabolic acid to maintain an acid-base homeostasis (51,52) and to promote calcium balance in bones (53). However, in the current study, dietary potassium was not associated with hip fracture risk in either gender (45). Further adjustment for potassium did not change the effect estimate for the relationship between carotenoids and hip fracture risk (data not shown). In our study, there was a null association between dietary vitamin C and hip fracture risk in either gender (data not shown). Although the Framingham Osteoporosis study showed that the use of supplementary vitamin C was associated with reduced fracture risk, there was no association with dietary vitamin C (54). Furthermore, the NHANES III study also showed no effects of dietary or serum ascorbic acid measures on bone mineral density and risk of self-reported fracture (55).

The novel finding of the current study is that the reduction in hip fracture risk associated with vegetables and carotenoids was most pronounced in lean men with BMI < 20 kg/m2. BMI is an established independent risk factor of osteoporosis from accumulative evidence (24,25), and appears to influence risk of hip fracture similarly between two genders (24). The proposed mechanism of a higher BMI as a protective factor against osteoporotic fractures has been attributed to greater weight-bearing in the loading bone sites for both genders (56), and increased endogenous estrogen produced in adipose tissues for postmenopausal women (57). However, our findings suggest that BMI had a greater influence on hip fracture development in men than in women. Low BMI has been associated with increased oxidative damage indexed by 8-hydroxy-2'-deoxyguanosine (27–29), particularly in men (28); and the molecule, 8-hydroxy-2'-deoxyguanosine, is considered a reliable biomarker for the measurement of systemic oxidative stress (58). Following this, we speculate that lean men possibly have higher oxidative stress in bones leading to hip fracture, and the antioxidant effects of carotenoids may counteract this mechanism of osteoporosis (e.g. the RANKL pathway) related to leanness. Another possible explanation is that since adipose tissue is the major storage site for carotenoids (59), the effects of insufficient carotenoid intake could be expected to be more apparent in lean subjects due to reduced storage and bioavailability.

The weaker association between BMI and hip fracture risk in women may be due to a possible non-differential misclassification of BMI, as some of the postmenopausal women in our cohort may experience weight gain after recruitment during menopause (60). In addition, it is plausible that the deleterious effect of a rapid decline of estrogen outweighs that of leanness related to oxidative stress on hip fracture development among postmenopausal women. If this is true, this is another possible explanation for the null association between vegetables/carotenoids and hip fracture risk in lean women <20 kg/m2. Taken together, our findings support the “estrogen-centric” role in postmenopausal osteoporosis and related fractures (26,61).

To our best knowledge, the current study is the first study to examine dietary carotenoids and hip fracture risk in an elderly Asian population. The strength of this study is the large number of incident hip fracture cases identified from a population-based prospective cohort with a long follow-up time. Since supplementation was relatively uncommon in this study population, nutrient consumption computed from dietary intake using the food frequency questionnaire was considered valid and relevant. Another strength is the presumed lack of recall bias in exposure data since they were obtained prior to disease diagnosis. Singapore is a small city-state with a system for easy access to specialized medical care. Since practically all hip fracture cases will seek medical attention immediately and be hospitalized, our case ascertainment through linkage with the comprehensive, nationwide hospital database can be considered complete. Finally, we included all established and other possible risk factors for hip fracture as covariates in our regression-based risk models to minimize the likelihood of spurious associations resulting from insufficient control of confounding. Limitations of this study include the possible misclassification of dietary intake from the use of a food-frequency questionnaire. However, such misclassifications are non-differential in nature, resulting in an underestimation (as opposed to an overestimation) of the true relative risk. Although the use of self-reported body weight and height could be prone to non-differential misclassification and thus lead to under-estimation of the BMI-hip fracture risk association, self-report of body weight has been shown to be highly valid across many populations (62) and specifically in Asians. A review of 64 studies suggested the difference between the self-reported and objectively measured body mass index was generally null or slightly underestimated (less than 1 kg/m2) for those with BMI less than 30kg/m2 (63), and 97% of our study population had BMI <30 kg/m2. Another limitation of this study is the lack of measurements on bone mineral density (BMD) at baseline, which may confound the relationship between BMI and risk of hip fracture, as BMD is closely related to body weight (64). Finally, we did not include any biological measurement for oxidative stress in our study, and were therefore unable to confirm our hypothesis.

In conclusion, the findings of the present study suggest that dietary vegetables and carotenoids exert a strong protection against hip fracture, especially in lean elderly men. Our epidemiologic findings are supported by experimental data that have provided the mechanistic pathway in the role of carotenoids in regulating the biology of osteoblasts and osteoclasts. Future research in mechanistic and intervention studies is warranted to evaluate the effect of carotenoids in relation to osteoporotic fractures among elderly.

Supplementary Material

Supp Material & Fig S1-S2

Acknowledgments

We thank Siew-Hong Low of the National University of Singapore for supervising the field work of the Singapore Chinese Health Study. We also thank the Ministry of Health in Singapore for assistance with the identification of hip fracture cases and mortality via database linkages. Finally, we acknowledge the founding, long-standing Principal Investigator of the Singapore Chinese Health Study – Mimi C Yu.

Funding: This study was supported by the National Medical Research Council, Singapore (NMRC/EDG/0011/2007) and National Institutes of Health, USA (NCI RO1 CA55069, R35 CA53890, R01 CA80205, and R01 CA144034).

Authors' roles: Study design: Z Dai and WP Koh. Study conduct and data collection: WP Koh, LW Ang and JM Yuan. Data analysis: Z Dai and RW Wang. Data interpretation: Z Dai, YL Low, JM Yuan and WP Koh. Drafting manuscript: Z Dai and WP Koh. Revising manuscript content and approving final version of manuscript: all authors. WP Koh takes responsibility for the integrity of the data analysis.

Footnotes

Disclosure: All authors state that they have no conflict of interest.

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