Subjects
This cross-sectional evaluation included 97 currently non-smoking, Caucasian, postmenopausal women free of chronic diseases (including severe osteoporosis) and medications (including estrogen) known to affect bone. All subjects reported at least 12 years of education (high school graduate), with many having additional college education. Subjects were part of a larger longitudinal study investigating the effects of a reduced sodium intake on bone, as described previously [20] and were randomly asked to participate in this study. Subjects were instructed to maintain a calcium intake of 1200 mg a day, the current recommended adequate intake [21], and were given calcium citrate supplements, if necessary, to assure adequate calcium intake throughout the study (as per the protocol of a larger study). All data for this study were collected by one person, a registered dietitian (RAB). The Institutional Human Subjects Review Board approved study protocol and subjects signed informed consent.
Anthropometry and bone densitometry
Weight and height were measured by standard procedures in indoor clothes without shoes. BMD (g/cm2) was measured by dual X-ray absorptiometry with a Lunar DPX-MD instrument (GE Medical Systems, Madison, WI, USA) using specialized software for whole body, lumbar spine, femur (neck, trochanter, Ward's triangle and shaft) and forearm (including radius and ulna), as described previously [22]. The BMD and anthropometries, were measured every 6 months throughout the study, but only those obtained at the time when cognition test was given (usually in a second year of the study) were used in analyses and in relation to cognitive status. Quality assurance of our densitometer was performed daily and coefficients of variation and precision of the instrument were reported previously [22].
Cognitive assessment
Cognition was assessed using the Mini Mental State Examination (MMSE) [23]. The MMSE is a brief, commonly used screening tool to assess cognitive status. It consists of various graded questions and tasks generating a maximum of 30 possible points. Each subject was asked a series of questions from eleven categories; orientation to time, orientation to place, registration, attention and calculation, recall, naming, repetition, comprehension, reading, writing and drawing. Scores are classified as: normal cognitive function with a score of 27–30; mild cognitive impairment with a score of 21–26; moderate cognitive impairment with a score of 11–20; and severe cognitive impairment with a score of 0–10 [24]. Cognitive function for each subject was assessed one time, usually in the second year of the study, and evaluated with the corresponding bone and anthropometric measurements conducted at that time, or with the cumulative average of other variables of interest.
Dietary and alcohol consumption assessment
Subjects were instructed by a registered dietitian to record 3 days of dietary intake (2 week and 1 weekend day) every 6 months during the study. Food models and pictures were used for instruction. After the records were completed, the same dietitian followed-up and rechecked every record with each participant, particularly about additional food or snacks consumed, portion sizes, and ways of preparation. The same dietitian analyzed nutrient intake from the records using Food Processor® (ESHA Research, Salem, OR). Mean daily intake, including total energy and all other macro- and micronutrients (vitamins, minerals and fatty acids) was calculated. Supplement use was recorded during each visit, as well. The detailed description of diet assessment is reported earlier [20]. Cumulative average intake of each considered nutrient, to the point of the cognition assessment (usually in a second year), was used to assess its relationship with cognitive functioning. Alcohol consumption was assessed using questionnaire designed to determine long-term (at least for a last year and extending back to several years), frequency, amount, and source of intake, with the help of the same dietitian. It was expressed as drinks per day, from which g/day of alcohol was calculated, as described previously [25].
Physical activity
Physical activity (PA) was assessed using the modified Allied Dunbar National Fitness Survey for older adults [26] and was collected every 6 months. A measure of total activity was assessed based on minutes per week engaged in weight bearing activities of a moderate intensity defined as an activity of at least 4 kcal/min, such as recreational activities (tennis, hiking, weight lifting), walking, heavy housework, gardening and do-it-yourself activities. Data collected included frequency and duration of each activity and were expressed as number of hours per week engaged in the above activities. The questionnaire was filled with the participants on the site and with the help of a dietitian. We used this questionnaire in our previous studies in postmenopausal women and found it reliable and easy to complete [27]. Cumulative average activity score to the point of the cognitive assessment was used in the subsequent analyses.
Data analysis
Data analysis was conducted with SPSS statistical software (version 8.0). Pearson's r was calculated among all variables, as a preliminary analysis. To determine the relationship between cognitive function and BMD, subjects were divided into two groups based on the mean (27.9) for the MMSE score. Group 1 comprised of subjects below and group 2 of subjects above the mean. One-way Analysis of Variance (ANOVA) was conducted for various bone sites and covariates to determine if there were significant group differences. Multiple Analysis of Covariance (MANCOVA) with univariate follow-up tests and Bonferroni corrections to the alpha level were used to check for group differences in BMD of the whole body, all sites of the hip (neck, trochanter, Ward's triangle and shaft), lumbar spine and forearm. Subjects' age, height, weight, hours of total activity, alcohol consumption and total energy, calcium and sodium intake were included in the analysis as confounders. The use of these confounders was based on scientifically proven and theoretically presumed evidence for their possible effects on bones and memory.
To determine the relationship between cognitive function and diet, subjects were divided into two groups based on MMSE criteria for normal cognitive function (score of ≥27). Group 1 (<27), was considered to have mild cognitive impairment while Group 2 (≥27), was considered to have normal cognitive function. Multiple regression models were calculated to find the best model using various nutrients to predict cognitive function. ANOVA was conducted for all nutrients and covariates. MANCOVA with confounders (described above) and univariate follow-up tests were used to check for group differences in nutrient intake. Significance level was set at P ≤ 0.05.
Declaration of sources of funding
This work was funded in part by the NRI/USDA 2001-00836, Donaghue Medical Research Foundation DF98-056, University of Connecticut Office for Sponsored Programs and Mission Pharmacal®, San Antonio, TX, USA. The financial sponsors played no role in the design, execution, analysis and interpretation of data, or in writing the study.