Association Between Raised Inflammatory Markers and Cognitive Decline in Elderly People With Type 2 Diabetes

RESEARCH DESIGN AND METHODS

The ET2DS is a sample of men and women aged 60–75 years with type 2 diabetes. The subjects were randomly selected by sex and 5-year age bands from the comprehensive Lothian Diabetes Register (LDR) of people with type 2 diabetes living in Lothian, Scotland. A sample size of 1,000 was targeted to detect associations between baseline risk factors and both cognitive ability at baseline and cognitive change during subsequent follow-up. Exclusion criteria, applied at the time of physical examination, have been reported previously (5). All subjects provided written informed consent, and the study was approved by the Lothian Research Ethics Committee.

Clinical assessment.

Physical examinations were performed between August 2006 and August 2007 by trained researchers using standard operating procedures (5). Quality control measures included tests of inter- and intra-observer variability. A fasting venous blood sample was taken for measurement of inflammatory markers, total serum cholesterol, and plasma A1C. Following measurements of height and weight, a 12-lead electrocardiogram (ECG) was taken for subsequent Minnesota coding (http://www.epi.umn.edu/ecg/). After a 10-min rest in the supine position, systolic and diastolic blood pressure was measured in the right arm using a standard stethoscope and aneroid dial sphygmomanometer. Right and left brachial, posterior tibial, and dorsalis pedis systolic pressures were then taken using an aneroid sphygmomanometer and a Doppler probe. A self-administered questionnaire included questions on education; history of myocardial infarction (MI), stroke or angina; year of diabetes diagnosis; smoking; current medications; and the World Health Organization (WHO) chest pain questionnaire (6). Data were collected from the Information and Services Division of the National Health Service (NHS) Health Services Scotland on all medical and surgical discharges from Scottish hospitals since 1981 (Scottish Morbidity Record [SMR01] scheme [7]), and any ICD-10 codes (or the equivalent ICD-9 codes) indicating cardiovascular or cerebrovascular disease were extracted.

Cognitive assessment.

Cognitive assessment took place after a snack and confirmation that blood glucose was >4 mmol/l. Fluid cognitive ability was assessed using tests of: nonverbal memory and immediate and delayed verbal declarative memory (Faces and Family Pictures Subtest, Logical Memory subtest from the Wechsler Memory Scale III^U.K. [8]); working memory, nonverbal reasoning, and processing speed (Letter Number Sequencing, Matrix Reasoning, Digit Symbol Test from the Wechsler Adult Intelligence Scale-III [WAIS III^U.K.] [9]); executive function (Verbal Fluency Test[10]); and mental flexibility (Trail Making Test–Part B [11]). Vocabulary (crystallized intelligence) was measured using the combined Junior and Senior Mill Hill Vocabulary Scale (MHVS) synonyms (12). As results on vocabulary-based tests vary little with ageing, they can be used to approximate peak prior cognitive ability (13). Late-life cognition adjusted for vocabulary also correlates highly with actual cognitive change (14). The Hospital Anxiety and Depression Scale (15) was also administered because mood can affect cognitive test results.

Measurement of inflammatory markers.

Blood samples were processed at the research clinic, and plasma was stored at −40°C. Assays for plasma CRP, IL-6, and TNF-α were performed in the University Department of Medicine, Glasgow Royal Infirmary. CRP was assayed using a high-sensitivity immunonephelometric assay (16). TNF-α and IL-6 antigen levels were determined using high-sensitivity ELISA kits (R&D Systems, Oxon, U.K.).

Data analysis.

Information on the questionnaire and recording forms was checked by clinic staff. Two clerks independently entered the data onto an Access database. Errors identified by comparing the double data entries were resolved by referring to the paper records. Anonymized data on demographic and clinical variables from the LDR were used to compare the characteristics of study responders and nonresponders.

Ankle brachial index, a measure of subclinical atherosclerosis (17), was calculated by dividing the lowest of the ankle pressures by the higher of the arm pressures. Smoking was categorized as current, ex, or never-smoked, with anyone stopping within the past 6 months recategorized as a current smoker. Duration of diabetes was calculated to the nearest year by subtracting the self-reported year of diagnosis from the date of attendance at the research clinic. The highest self-reported level of education was categorized as primary, secondary, professional qualification, or university/college degree. Scottish Index of Multiple Deprivation (deprivation) (18) was assigned to each subject according to their postcode of residence. The following criteria were used to define MI: 1) subject recall of a doctor's diagnosis of MI, 2) positive WHO chest pain questionnaire for MI, 3) ECG evidence of ischemia (Minnesota codes 1.1–1.3, 4.1–4.2, 5.1–5.3 or 7.1), and 4) prior hospital discharge code for MI (ICD-10 codes I21–I23, I252). MI was recorded if two of the first three criteria were met or if both the first and last criteria were met. Equivalent criteria for angina were: 1) subject recall of a doctor's diagnosis of the condition or being on regular medication for angina, 2) positive WHO chest pain questionnaire for angina, 3) ECG evidence of ischemia, and 4) prior hospital discharge code for ischemic heart disease (ICD-10 codes I20–I25). Angina was recorded if two of the first three criteria were met or if both the first and last criteria were met. Stroke was recorded if two of three of the following criteria were met: 1) subject recall of a doctor's diagnosis of stroke, 2) prior hospital discharge code consistent with stroke (ICD-10 codes I61, I63–I66, I679, I694), and 3) confirmation by review of clinical notes that the event was not due to a transient ischemic attack.

Continuous variables were normally distributed apart from Trail Making Test scores, CRP, IL-6, and TNF-α levels, which were transformed using natural logarithms. The inflammatory markers were trimmed for points exceeding 3.5 SDs from the mean prior to analysis. Scores from the fluid cognitive tests were used to obtain a general cognitive ability factor (g) via principal components analysis.

Age- and sex-adjusted linear regression assessed the association between cognitive scores and inflammatory markers. Further adjustments were made for vocabulary (MHVS) and then for additional covariates to adjust for mood (Hospital Anxiety and Depression Scale depression score), duration of diabetes, glycemic control (plasma A1C), cardiovascular risk factors (total cholesterol, BMI, diastolic blood pressure, smoking), cardiovascular disease (MI, angina, stroke, ankle brachial index), and level of education. The analyses had over 90% power for a two-sided significance test (α = 0.05) to detect a standardized β coefficient of 0.10. Analyses were performed using R version 2.7.0 (19).