Subjects

The Framingham Study cohort has been evaluated biennially since 1948. Between 1976 and 1978, a total of 2611 subjects were enrolled in a dementia-free cohort.21,22 At the 20th biennial examination (between 1986 and 1990), 1592 subjects from this cohort were alive and free of dementia and had follow-up data for at least one year. Of these subjects, 1229 (77 percent) underwent the 20th examination, and in 1092 participants (89 percent of those examined), plasma total homocysteine levels were measured. These 1092 subjects constituted our study sample. There were 667 women and 425 men, and their mean (±SD) age was 76±6 years (range, 68 to 97). Informed consent was obtained from all study subjects with the use of a consent form approved by the institutional review board for human research at the Boston University School of Medicine.

Diagnosis of New Cases of Dementia and Alzheimer's Disease

Subjects in the cohort that was free of dementia at inception have been monitored with published surveillance techniques since 1978 for the development of stroke or dementia.21,22 Methods have included a screening Folstein Mini–Mental State Examination23 at each biennial evaluation, followed by annual neurologic and neuropsychological assessment of subjects with suspected cognitive impairment.

The final diagnosis of dementia was made by a committee, comprising at least two neurologists and a neuropsychologist, that determined the type of dementia and the date of diagnosis. All available information was used to evaluate participants with suspected dementia, including serial neurologic and neuropsychological assessments, a telephone interview with a family member or care giver, medical records, imaging studies, and autopsy data when available. The review committee was unaware of the subjects' plasma homocysteine levels. The diagnosis of dementia was made according to the criteria of the Diagnostic and Statistical Manual of Mental Disorders, fourth edition24; our definition also required a duration of symptoms greater than six months, and a score for severity of dementia of 1 or higher on the Clinical Dementia Rating scale.25 Alzheimer's disease was diagnosed when subjects met the criteria of the National Institute of Neurological and Communicative Disorders and Stroke and the Alzheimer's Disease and Related Disorders Association for definite, probable, or possible Alzheimer's disease.26

Plasma Homocysteine

Plasma total homocysteine levels were measured in all subjects at the 20th biennial examination (base line). An earlier measure from the 16th biennial examination (performed between 1979 and 1982, approximately eight years before base line) was also available for 935 of the subjects (86 percent). All plasma specimens were stored at or below –20°C. Homocysteine levels were determined with the use of high-performance liquid chromatography with fluorometric detection.27 The coefficient of variation for this assay was 9 percent.28

Apolipoprotein E Genotypes

Data on the apolipoprotein E (APOE) genotype were available for 1012 of the subjects (93 percent). The presence of particular alleles was determined by means of isoelectric focusing of the plasma and confirmed by DNA genotyping.29,30 Participants were divided into two groups, one comprising persons with an APOE ε4 allele (ε2/ε4, ε3/ε4, or ε4/ε4 genotype) and another comprising those without an APOE ε4 allele.

Vitamin Levels

Plasma concentrations of folate, cyanocobalamin (vitamin B 12 ), and pyridoxal-5'-phosphate (the coenzyme form of vitamin B 6 ) were estimated at the 20th biennial examination. Plasma folate was measured by a microbial (Lactobacillus casei) assay with a 96-well plate and manganese supplementation31; plasma vitamin B 12 levels were estimated with the use of a radioassay kit (Magic, Ciba–Corning, Medfield, Mass.); and pyridoxal-5'-phosphate was measured by the tyrosine decarboxylase apoenzyme method.32 Coefficients of variation for these assays were 13 percent for plasma folate, 7 percent for cyanocobalamin, and 16 percent for pyridoxal-5'-phosphate.28 Because of insufficient plasma samples, the vitamin levels were not determined for all patients. Of the subjects with measurements of plasma homocysteine, 85 percent had measurements of vitamin B 12 , 92 percent had measurements of vitamin B 6 , and 98 percent had measurements of folate.

Definition of Additional Risk Factors

Risk factors that could potentially confound the relation between plasma homocysteine and dementia or Alzheimer's disease were defined with the use of data collected at the 20th biennial examination. When appropriate, data from earlier biennial examinations were also used. Educational status was dichotomized at the level of high-school completion. We adjusted the analyses for cigarette smoking using two variables: current smoking status (smoker or nonsmoker) and lifetime exposure to cigarette smoke (<5.0 pack-years, 5.0 to 29.9 pack-years, or ≥30.0 pack-years). Alcohol intake was categorized in terms of the number of drinks per day: zero, less than one, one to two, or more than two.33 Diabetes mellitus was defined by a recorded casual blood glucose level of at least 200 mg per deciliter (11.1 mmol per liter), a previous diagnosis of diabetes mellitus, or the use of a hypoglycemic agent or insulin. Systolic blood pressure and body-mass index (the weight in kilograms divided by the square of the height in meters) were treated as continuous variables.

Statistical Analysis

The distribution of plasma homocysteine levels in the population was positively skewed. The use of natural-log–transformed values provided the best-fitting model for analyses in which the plasma homocysteine level was treated as a continuous variable. Plasma homocysteine levels were also evaluated with a quartile-based analysis. Since homocysteine levels increase markedly with age,28,34,35 the quartiles were defined in an age-specific manner for each of several five-year age categories.

Cox proportional-hazards regression models36 were used to examine the relation between the homocysteine level and the incidence of dementia and Alzheimer's disease during follow-up, after adjustment for age (in one-year increments), sex, and APOE genotype (with or without an APOE ε4 allele).37 In supplementary analyses, we also adjusted for vitamin levels and other covariates. Subjects were followed for new cases of dementia from the date of their 20th biennial examination until December 31, 2000. For the analysis of new cases of Alzheimer's disease, data for subjects in whom other types of dementia developed were censored at the date of the diagnosis of dementia, since the diagnostic categories were mutually exclusive. Subjects who had a stroke during the study period were not excluded, since such an event could be part of the causal chain between an elevated plasma homocysteine level and the development of dementia. All statistical analyses were performed with the use of SAS software (SAS Institute, Cary, N.C.).