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3 Centre for Ageing and Public Health, London School of Hygiene and Tropical Medicine, London WC1E 7HT, UK; 4 Department of Epidemiology and Public Health, University College London, London WC1E 6BT, UK; 5 Clinical Trial Service Unit, Nuffield Department of Clinical Medicine, Oxford OX3 7LF, UK; and 6 Institute of Human Nutrition, University of Southampton Medical School, Southampton SO16 6YD, UK
* To whom correspondence should be addressed. E-mail: alan.dangour{at}lshtm.ac.uk.
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ABSTRACT |
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 TOP ABSTRACT IntroductionSubjects and MethodsResultsDiscussionLITERATURE CITED |
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75 y living in the United Kingdom. In a population-based prospective cohort study, 853 men and women aged 75 y were examined in 1995–98 as part of the Medical Research Council Trial of Assessment and Management of Older People in the Community. During a median follow-up of 7.6 y (5528 person-years of follow-up), 429 individuals (50.3%) died, including 185 from CVD. Individuals with plasma homocysteine levels in the top one-third compared with the bottom one-third had a 2-fold higher risk of all-cause mortality (hazard ratio, 2.20; 95% CI, 1.76, 2.75; P < 0.001) and CVD mortality (hazard ratio, 1.96; 95% CI, 1.39, 2.78; P < 0.001) after adjustment for age, sex, and other covariates. There was no association of plasma folate or vitamin B-12 levels with mortality. Our results extend previously reported associations of homocysteine with mortality, and the absence of associations of folate and vitamin B-12 with mortality, to the older population.
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Introduction |
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TOPABSTRACTIntroductionSubjects and MethodsResultsDiscussionLITERATURE CITED |
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There is, however, very limited information on the relevance of elevated homocysteine levels for CHD and stroke risk in older people. Moreover, whereas the evidence for the associations of folate or vitamin B-12 status with cardiovascular disease (CVD) or CVD mortality in middle-age individuals remains inconclusive (5–13), it is even more inconsistent and limited among older people (14–17). The "homocysteine-hypothesis" of vascular disease has attracted considerable interest, because homocysteine can be easily lowered by folic acid and vitamin B-12 supplementation, raising the prospect that dietary intervention with B-vitamins could lower the risk of CHD and stroke (18).
Homocysteine metabolism is frequently compromised in older individuals in whom low vitamin B-12 status, due to vitamin B-12 malabsorption caused by an age-related decrease in gastric acid production or gastric atrophy leading to poor production of intrinsic factor, is common (19,20). The metabolism of homocysteine links the methionine transsulfuration pathway with the folate cycle; hence, adequate intake of the B vitamins, folate and vitamin B-12, is required to avoid hyperhomocysteinemia. Plasma homocysteine levels are also influenced by renal function, which declines with advancing age and is an independent risk factor for coronary disease (21). These age-related alterations in B vitamin status may influence the putative associations with burden of disease and death from CVD in later life.
Dietary supplementation with both folic acid and vitamin B-12 typically reduces plasma homocysteine concentrations by 25–30% in populations consuming unfortified foods (22,23). The possibility that folate and vitamin B-12 supplementation could reduce mortality has thus attracted considerable interest in view of the widespread use of B vitamin supplements and the national policies of mandatory fortification of flour with folate. However, concern exists about the possible adverse effects of folic acid supplementation (24–26), particularly in older individuals with low vitamin B-12 status (27).
The aim of this study was to investigate the associations of plasma levels of folate, vitamin B-12, and homocysteine with death from all causes and from CVD in a large cohort of older people (aged 75–84 y at baseline) living in the United Kingdom, where folic acid fortification is voluntary.
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Subjects and Methods |
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TOPABSTRACTIntroductionSubjects and MethodsResultsDiscussionLITERATURE CITED |
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75 y who were registered with a general practitioner were invited to participate in the trial unless they were a resident in a long-stay hospital or nursing home or were terminally ill (28). In-depth trial assessments included questionnaires seeking information about aspects of health (30), alcohol intake and smoking habits, sociodemographic factors (including marital status, living circumstances, and housing tenure), and medical history, including past and recent history of heart attack, stroke, cancer, and diabetes. Two repeat measures of blood pressure (sitting and standing), height (with use of a stadiometer), and weight (using Soehnle scales; Leifheit AG) were recorded. Participants were registered for mortality follow-up with the UK government's Office for National Statistics.
The nutrition study was designed as a substudy within the trial to examine the associations between blood levels of antioxidant vitamins, lipids, and folate with mortality, including deaths from CVD. As well as measuring blood levels of micronutrients, dietary intake information was collected. The nutrition and physical activity interview, and the nutrition study blood results, were not part of the trial interventions and participants were not provided with any information or advice regarding their blood results or diet. Written informed consent was obtained from the participants and the main trial and substudy were approved by the 53 local research ethics committees responsible for each of the participating general practices.
Sample size and selection. General practitioners in the 53 practices in the trial undertaking universal screening were invited to take part in the nutrition substudy. A sample size for the nutrition study of 3000 individuals (with an expected response rate of 70%) was chosen to provide 80% power at a 5% significance level to detect a protective effect of 0.6 between the highest and the lowest quintile of B vitamin distribution, assuming a mortality rate of 60/1000 person-years (py). The participant age group was restricted to 75–84 y at baseline to avoid participant burden in the older age categories. For each participating general practice, persons with birth dates within the eligible range were identified from the age-sex registers and a systematic random sample was taken by applying a predetermined sampling fraction, which selected a similar number of participants for each practice. At the trial in-depth assessment, the nurse invited the selected participants to take part in a further interview (to be held 4 wk after the assessment) and to provide an additional 8 mL of blood. To take account of seasonal variations in diet that might influence blood vitamin concentrations, the invitation dates were evenly spread across the year; however, a higher percentage of blood was taken in the period March to June (40%) than in the periods July to October or November to February (both 30%).
Blood collection. Blood was collected in nonfasting conditions under subdued lighting into 7- or 9-mL EDTA-containing tubes, which were placed immediately into an ice pack to be stored in a domestic refrigerator at 4°C until shipment on ice in an insulated container to a local laboratory within 4 h of collection. At the laboratory, the blood was immediately centrifuged at 1000 g for 10 min at 4°C, and the plasma was then divided into 7 aliquots and frozen to –80°C. The local laboratories stored the blood samples for a maximum of 6 mo until they were sent in batches by 24-h courier on dry ice to the Rowett Research Laboratories (Aberdeen, UK) for further storage at –70°C.
Plasma vitamin B-12 and folate concentrations were measured at the Rowett Laboratory using a Becton Dickinson Simultrac kit for vitamin B-12 and folate (31), with a CV of 12–15% for vitamin B-12 and 9–12% for folate. Plasma homocysteine was measured at the University Department of Pharmacology in Oxford using a fluorescence polarization immunoassay (AXIS-Shield) on an Abbott IMx auto-analyzer (32) with a CV of <3.5% (33). Total and HDL cholesterol were determined using an automated spectrophotometric clinical analyser (Kone Dynamic, Kone Intruments).
As part of the nurse assessment, a nonfasting blood sample and midstream urine sample were also taken to assess kidney function. Of the 45 local laboratories to which samples were sent, 37 used the modified Jaffe method for serum creatinine, 7 used an enzymatic method, and in one the method was unknown. The simplified modification of diet in renal disease method was used to estimate glomerular filtration rate (eGFR) (34,35), which was then categorized into 3 groups with corresponding chronic kidney disease (CKD) stages (36) (CKD stages 3 and 4 were combined, because only 1.2% of participants had very poor kidney function).
Interviews. The nutrition and physical activity interviews were carried out by the UK government's Office for National Statistics Social Survey Division and took place after the blood collection (median gap of 27 d). The nutrition interview used the UK European Prospective Investigation into Cancer and Nutrition study version of a FFQ originally developed by Willett et al. (37), with some minor modifications. The questionnaire included 139 food groups. In each case, participants indicated their frequency of consumption of food groups over the previous year by choosing from responses coded 1 to 9. Estimates of portion sizes were based on the equivalent age group from the National Diet and Nutrition Survey (38). Intake of liver, lamb, fish, and dark green vegetables were chosen for inclusion in our analysis as rich sources of B vitamins. Information on supplement use and type was also collected but was not added to daily intake because of lack of data on dosage. No appropriate physical activity questionnaire was available for this age group in the UK and we therefore adapted questions from the Allied Dunbar National Fitness Survey to cover a range of activities, including leisure activities, housework, and home maintenance, which were further categorized according to time spent and intensity of effort (39).
Mortality. The Office for National Statistics provided the date and coded cause of death using the International Classification of Diseases (ICD) 9th revision (ICD-9) for deaths reported up to September 2002 (43% of deaths in this analysis) or ICD-10 after that date and up to August 2006. CVD was classified as a death from an underlying cause (ICD-9 390–459 or ICD-10 I1-I99).
Statistical analysis. To allow for adjustment for potential confounders, analyses were performed for respondents with complete data on health assessments, nutrition interviews, and blood results. The results presented are weighted to allow for differential selection in the individual practices. Participants were grouped according to thirds of plasma levels of folate, total homocysteine, and vitamin B-12. Age, anthropometry, biochemical variables, alcohol consumption, and physical activity variables were treated as continuous data. All participants who reported regular use of any supplements were categorized as ‘supplement users.’ The food frequency variables used in the analysis (consumption of liver, lamb, fish, and green vegetables) were treated as categorical variables as follows: liver at least once every 2 wk vs. less frequently; lamb at least once a week vs. less frequently; fish at least twice a week vs. less frequently; green vegetables more than once a week vs. less frequently. History of CVD, cancer, or diabetes, living alone and housing tenure (owner occupier vs. not) were binary variables. Participants were classified as current smokers, ex-smokers, or nonsmokers.
Hazard ratios for mortality were estimated for thirds of plasma levels of folate, total homocysteine, and vitamin B-12 referent to the lowest third, respectively, using Cox regression models and modified Wald tests for significance. All analyses took account of the cluster design in the estimation of 95% CI (40). All models were adjusted for age and sex. The P-values for test for trend were obtained from a logistic regression model in which the thirds were scored from 1 to 3 and a log-linear model for odds of the outcome assumed. Further models took account of possible confounders from data collected at the in-depth assessment and the nutrition and physical activity interviews. The final models were adjusted only for those factors that remained associated with at least 1 of the outcomes (P < 0.05) in the presence of the other factors. Analyses were performed using Stata 8 software (41).
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Results |
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TOPABSTRACTIntroductionSubjects and MethodsResultsDiscussionLITERATURE CITED |
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The sample (n = 853; mean age at baseline, 78.6 y) was similar to both the group that had undergone an in-depth assessment in the main trial and were sampled to take part in this nutrition study and to all eligible individuals in the main trial (Table 1). The group used in our analysis was younger, contained a slightly greater proportion of men, and was less likely to have reported a history of cancer. The median follow-up time after the baseline interview was 7.64 y (interquartile range, 4.51–8.51 y; maximum of 10.24 y) and by the end of August 2006, 429 people in the sample had died, 185 (43%) of which were from CVD. Deaths from all causes and from CVD alone suggest a pattern for increased mortality with increased homocysteine level but not with either folate or vitamin B-12 status (Table 2).
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1.7 times higher in the middle third and 2.2 times higher in the top third of homocysteine compared with the bottom third. Vitamin B-12 levels showed no association with either all cause or CVD mortality (Table 6).
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Plasma homocysteine, but not folate or vitamin B-12, was strongly associated with eGFR. However, inclusion of CKD stage in the models did not significantly alter the hazard ratios presented in Table 6 (data not shown). There were also no significant interactions with CKD stage for either folate or vitamin B-12 and mortality.
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Discussion |
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TOPABSTRACTIntroductionSubjects and MethodsResultsDiscussionLITERATURE CITED |
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75 y in the UK. Although renal function was associated with homocysteine level, it did not explain the association of homocysteine with mortality. Plasma folate and vitamin B-12 were not associated with all-cause mortality in the setting of voluntary fortification. It is possible, for folate and vitamin B-12, that this study was based on too few events and lacked the statistical power to detect a modest association with CVD and all-cause mortality. The 95% CI for the hazard ratio indicates that the present study could exclude effects greater than 1.58 for folate and 1.33 for vitamin B-12. Moreover, no associations were detected when these nutrients were categorized into thirds or when treated as continuous variables and no interaction between folate and vitamin B-12 was detected in the relationship with mortality.
Although evidence for the existence of an association of folate or of vitamin B-12 with all-cause or CVD mortality is inconsistent, the association of homocysteine with mortality is well established, at least among younger adults. A meta-analysis of 72 studies (mean subject age 55 y), both genetic and prospective, into the association between plasma homocysteine and ischemic heart disease, deep vein thrombosis, and pulmonary embolism has provided support for a causal association (4). Our study similarly found a significant increased risk of all-cause and CVD mortality across all levels of plasma homocysteine concentration even in adults aged 75 y at baseline.
Two studies have provided weak evidence that low folate concentrations are associated with increased risk of death, although neither found evidence of a gradual increase in risk with declining folate but instead suggested a threshold effect with only those with the very lowest folate concentrations being at high risk (8,14). More studies have looked specifically at CVD mortality in relation to folate levels, but the picture here is also inconclusive. Some studies have found significant negative associations, with lower folate concentrations being associated with higher risk of CVD mortality (6,11,12,16), whereas others have found no evidence of an association of folate with CVD or CVD mortality (5,7,9,10,15,17). The longest study to date (2950 adults followed for 29 y) showed no association between folate and CVD mortality but was based on adults aged 20–90 at baseline (mean age 48 y) (9). The null results of many of the North American studies of folate and CVD mortality may reflect greater use of multivitamin supplements and folic acid fortification in North America (5,7,10,17). The much more limited data on vitamin B-12 concentration and mortality risk suggested no association between vitamin B-12 concentration and CHD or CVD mortality in 2 studies (9,13), a negative association with vascular disease mortality in 1 study (15), and a positive association with all cause mortality in the remaining study (17). Our analysis adds to the literature in finding no association between plasma vitamin B-12 concentration and mortality even in this older cohort aged 75 y at baseline.
The mean plasma folate, homocysteine, and vitamin B-12 concentrations in our study were similar to those found for participants aged 75 y in 2 other UK-based studies: the Oxford Healthy Aging Project and the National Diet and Nutrition Survey (42). The mean plasma folate concentrations are slightly higher in our current study than in either of the latter studies, possibly due to improvements in folate status resulting from voluntary fortification in the UK population.
Measurements of folate, vitamin B-12, and homocysteine were based on a single nonfasting blood sample and this is likely to underestimate the associations with mortality due to regression dilution bias. A previous study of the within-person variability in plasma levels of homocysteine reported intraclass correlation coefficients of 0.89 for homocysteine, 0.84 for vitamin B-12, and 0.81 for folate [(43) and unpublished data]. Similarly, for eGFR, the use of a single blood sample may have resulted in some residual confounding in our analysis including CKD. There is considerable variability between assays and between laboratories in the measurement of folate (44–46), although this will likely not affect the associations presented in this study, which were all conducted in the same laboratory. Finally, although the stability of homocysteine determinations may have been impaired by the delay in separation of plasma from red cells, the samples in this study were stored at 4°C, which should minimize the release of homocysteine from red cells (47).
As anticipated (42), in our study, plasma folate and vitamin B-12 concentrations were inversely associated with homocysteine concentration, and plasma folate and vitamin B-12 concentrations were positively associated with each other. In general, high plasma folate and vitamin B-12 concentrations and low homocysteine concentrations in our study were related to features of good heath or health-seeking behavior, consistent with the literature (8,12,14,16,42). Several large randomized trials are currently assessing the relevance of lowering homocysteine levels for prevention of cardiovascular events and the results of the totality of the available evidence should guide public health policy on using folic acid supplements or folic acid fortification for the prevention of cardiovascular events (48).
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FOOTNOTES |
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2 Author disclosures: A. D. Dangour, E. Breeze, R. Clarke, P. S. Shetty, R. Uauy, and A. E. Fletcher, no conflicts of interest.
7 Abbreviations used: CHD, coronary heart disease; CKD, chronic kidney disease; CVD, cardiovascular disease; eGFR, estimated glomerular filtration rate; ICD, International Classification of Diseases; MTHFR, methylene-tetrahydrofolate reductase; py, person-years.
Manuscript received 11 October 2007. Initial review completed 4 December 2007. Revision accepted 12 March 2008.
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