ULSAM-82 Methods

Demography

The age was calculated as  (examination date-birth date)/365.25 and presented with one decimal.

Anthropometry

Height was measured to the nearest whole 0.5 cm, and  body weight to the nearest 0.1 kg. The body mass index  (BMI) was calculated as the ratio of the weight (in kg) to the height (in meters) squared (kg/m²). The  waist and hip circumferences were measured in the supine position. The  waist was measured midway between the lowest rib and the iliac crest and the  hip over the widest part. The waist/hip ratio was calculated.
Sagittal abdomen diameter (antero-posterior) was measured with ptients in underwear and in a recumbent position with hips fixed, on a firm examination table. At the level of the iliac crest, the perpendicular distance between the examination table up to the horizontal level was measured during a normal expiration to the nearest 0.1 cm. 
Over arm circumference was measured at the midsection of the brachial point of the arm with the arm alongside in the supine position.
Calf circumference was measured at the midsection of the crus, between the knee joint and the ankle joint (exactly between the patellar bone midpoint and the most protruding part of the medial malleol).

Office blood pressure

Blood pressure was measured in the right arm with the subject in the supine position after resting for 10 minutes, and then after the subject had been standing for 2 minutes. The values was recorded twice and to the nearest even figure. The means of the two values are given for each blood pressure. The cuff size was 12·35 cm or 15·45 cm depending on the arm circumference. Systolic and diastolic blood pressures were defined as Korotkoff phases I and V, respectively.

Glucose and insulin

Plasma glucose concentrations were measured by the glucose dehydrogenase method (Gluc-DH, Merck, Darmstadt, Germany).

Plasma insulin was assayed by using an enzymatic-immunological assay (Mercodia Insulin ELISA) performed in a CODA Automated Analyser (BIO-RAD) and was given in mU/l.

Chemical lab

HBA1C (B-hemoglobin A1c) was measured at Uppsala Akademiska Sjukhus using a high pressure liquid chromatograph with a gradient system (BIO-RAD Laboratories). The method measures HbA1c in the range 1.3-18.9%. The reference interval is 3.9-5.3% for men and women above 50 years of age. The HbA1c mirrors the mean blood glucose level during the last 2 to 6 weeks before the time of sampling.

Cystatin C Biomarker of renal dysfunction. Serum cystatin C measurements were performed by latex enhanced reagent (N Latex Cystatin C, Dade Behring, Deerfield, IL, USA) using a Behring BN ProSpec analyzer (Dade Behring). The total analytical imprecision of the method was 4.8 % at 0.56 mg/L and 3.7% at 2.85 mg/L. 
GFR in mL/min can be calculated from serum cystatin C results in mg/L by the formula y = 77.24x-1.2623. The assays were performed at the Department of Clinical Chemistry, University Hospital, Uppsala, which is ackreditated according to 17025 by Swedac.

Interleukin-6 (IL-6) High sensitivity IL-6 was analyzed by an ELISA kit (IL-6 HS, R&D Systems, Minneapolis, MN). Samples and standards were pipetted in a microtiter plate coated with monoclonal antibody against IL-6. After incubation and washing enzyme substrate solution was pipetted and followed by anti-IL-6 antibody. The color reaction was proportional to the bound IL-6. The total CV of the method was 7 % and interassay CV was 5%.

C-Reactive Protein (CRP) An acute phase protein associated with inflammation and infection. High sensitivity CRP measurements were performed by latex enhanced reagent (Dade Behring, Deerfield, IL) using a Behring BN ProSpec analyzer (Dade Behring). The intraassay CV of the CRP method was 1.4 % at both 1.23 mg/L and 5.49 mg/L.

Serum Amyloid protein A (SAA) An acute phase protein associated with inflammation and infection. SAA measurements were performed by latex enhanced reagent (Dade Behring, Deerfield, IL) using a Behring BN ProSpec analyzer (Dade Behring). The intraassay CV of the SAA method was 5.9 % at 12.8 mg/L and 3.2 % at 81.7 mg/L.

Ab40 and Ab42 in EDTA-plasma were analysed by enzyme-linked immunosorbent assay (ELISA) using previously described method (Fukumoto et al. 2003). To block cross-reaction of unidentified components of human plasma with the ELISA, plasma was precleared with mouse IgG1 (Sigma-Aldrich Corp, St Louis, Mo) cross-linked to agarose beads (CNBr-activated Sepharose 4B; Amersham Biosciences, Piscataway, NJ). For the capture of Ab40 and Ab42 were used BNT77 antibodies (mouse IgA anti-A 11-28; Takeda Chemical Industries, Osaka, Japan) and for the detection peroxidase-conjugated detector antibodies BA27 (mouse IgG2 anti-Ab40, Takeda Chemical Industries) and BC05 (mouse IgG1 anti-Ab42, Takeda Chemical Industries) for Ab40 and Ab42 respectively. Ab40 was analyzed in duplicate and Ab42 in triplicate samples using identical lots of reagents. Reference: Fukumoto H, Tennis M, Locascio JJ, Hyman BT, Growdon JH and Irizarry MC (2003). Age but not diagnosis is the main predictor of plasma amyloid beta-protein levels. Arch Neurol 60: 958-964.

Lipids

Cholesterol and triglyceride concentrations were analysed in serum and isolated lipoprotein fractions by enzymatic techniques using Konelab Triglycerides ref 981786 and Konelab Cholesteol ref 981813 for use in a Konelab 20 Clinical Chemistry Analyzer (Thermo Electron Corporation, Vantaa, Finland).
HDL particles were separated by precipitation with magnesium chloride and phosphotungstate.
LDL cholesterol was calculated using Friedewald's formula: LDL=serum cholesterol-HDL-(0.45·serum triglycerides).

Cognitive function

The mini mental state examination (MMSE) ( Folstein 1975) is a screening test for dementia and cognitive decline, which is widely used both in clinical practice and in research. It has the advantage of being easy to administer, and it has a high replicability. On the other hand, it is insensitive to minor cognitive dysfunction.

The 7 minute screen test) is a another screening test for dementia and cognitive decline.
We used a Swedish translation of the 7MS which consists of four brief cognitive tests:
Benton temporal orientation: In this test the orientation in time is measured and quantified in degree of error. The maximum score is 113 (10 error points for a year, 5 points for a month, 1 for the date and the day of a week, and 1 for each 30 minute deviation in time). 
Enhanced cued recall: The subject has to identify 16 pictures, which are recalled immediately and after a brief interval. The investigator gives a semantic cue (for example an eagle is cued as a bird). The subject has to name and memorise four pictures at a time. When all four pictures are successfully recalled (the subject may see them twice), another set of pictures is shown. After a short period in which another task is presented, the subject is asked to recall all pictures. The investigator gives the respective semantic cues for pictures, which are not spontaneously recalled by the subject. The total score (maximum 16) is the number of pictures, remembered either freely or after a cue is given.
Clock drawing: In this widely used cognitive test the subject has to draw the face of a clock and place the hands of the clock at a fixed time. The maximum score is 7 points.
Verbal fluency: The subject has to name as many different animals as possible in one minute time. There is a maximum score of 45. 
(Reference: Solomon PR, Hirschoff A, Kelly B et al: "7 minute neurocognitive screening battery highly sensitive to Alzheimers's disease". Arch Neurol 1998;55:349-55)

Diseases and drugs

Information on what drugs the participants were taking was collected on a separate form. The definitions of prevalent diseases were made according to the definitions used in previous ULSAM investigations and not according to the present guidelines.
Hypertension prevalence (W102) was defined as diastolic blood pressure (W014) greater or equal to 95 mmHg and/or treatment with anti-hypertensive drugs for hypertension. Men treated with these drugs for other reasons, i.e. cardiac failure, are thus not included in this definition.
Hyperlipidaemia prevalence (W103) was defined as serum cholesterol levels (W972) above 6.5 mmol/l and/or serum triglyceride levels (W791) above 2.3 mmol/l and/or use of lipid medicines (W103).
Diabetes prevalence (W378) was defined as fasting plasma glucose (W319) of 7.0 mmol/l or above and/or use of diabetes medicine (W405).

ECG & Minnesota codes

A standard 12-lead ECG was recorded at 50 mm/s and 10 mm/mV and evaluated according to the Minnesota code ( Blackburn et al 1960 and in revised form in  Prineas et al 1982) by one experienced physician who was unaware of other data of the subjects. The Minnesota Code is the most widely used ECG coding scheme in epidemiological practice. 
ECG status was classified as normal or abnormal on the basis of presence of one or more of the ECG diagnoses listed below.

ECG diagnosis Minnesota code
Q or QS pattern 1.1
Q or QS pattern 1.2 or 1.3
High amplitude R waves 3.1 or 3.3
ST junction and segment depression 4.1 or 4.2
T-wave items 5.1 or 5.2 or 5.3
AV conduction defects 6.X
Left bundle branch block 7.1
Ventricular conduction defects 7.X
Atrial fibrillation or flutter 8.3
Arrhythmias 8.X
Artificial pacemaker 6.8

DXA

BDUAL-ENERGY X-RAY ABSORPTIOMETRY (DXA) Of the 530 participants in the final investigation, 507 agreed to undergo measurements of the bone mineral density (BMD, g/cm2) and bone area (cm2) of the total body, femoral neck region of the hip, total proximal femur, total legs and arms, the skull, and the lumbar spine (vertebrae L2-L4), as well as total lean and fat mass, by DXA (DPX Prodigy, Lunar corp., Madison, WI, USA). When applicable, both extremities were used in the calculation. By triple measurements in 15 subjects, the precision error of the DXA measurements in our laboratory has been calculated to be between 0.8 and 1.5% for BMD depending on site, and between 0.7 and 1.6% for bone areas. Total fat mass had a precision error of 1.5% and total lean mass 1.0%.

VITAMINS

Vitamin A was analysed according to the ref. " Quantitative high-throughput determination of endogenous retinoids in human plasma using triple-stage liquid chromatography/tandem mass spectrometry".
Thomas E. Gundersen, Nasser E. Bastani and Rune Blomhoff.
Rapid Communications in Mass Spectrometry. Rapid Commun. Mass Spectrom. 2007; 21: 1176-1186 
Published online in Wiley Interscience (www.interscience.wiley.com) DOI: 10.1002/rcm.2946 

Vitamin K was analysed

Dietary assessment

Dietary intake was assessed with a seven-day food record questionnaire and 257 of the participants completed the seven day food recording.
A precoded 7-d record book was used. This book was developed at the the Swedish National Food Administration, (NFA) Livsmedelsverket. (The method is described in detail in Riksmaten 1977 -98 Kostvanor och näringsintag I Sverige, Metod- och resultatanalys Livsmedelsverket 2002 in Swedish, short summary in English).
The pre-coded record book gives pre-printed alternatives (with quantity indications in household measures) for foods, meal components and facilities for indication of where and when the meals are consumed) Using a portion-guide with photographs, different sizes of cooked food portions and salads eaten at main meals could be estimated. The use of fat spreads on sandwiches was estimated with the help of photographs in a similar way. For snacks and other in-between meal eating was recorded in household measures, pieces, etc., using pre-coded alternatives. Additional consumption of food and drink was recorded in free text.
Data input and dietary analysis was performed using a commercial software package (MATs), which included food composition data from the NFA (PC-kost 4.03). The pre-printed alternatives were directly converted into amounts and NFA food codes.Only for foods recorded in free text coding by dieticians were needed prior to data input.
All participants were informed and instructed about the dietary survey by two dieticians. In order to collect the data and check that all the questions were answered the dietician visited, if agreed, the participant in the participant´s home. If the participant had not or was not able to complete the investigation for instance poor eyesight the dietician helped to fill in the records. If the participants did not agree to a visit by the dietician they could mail the seven-day food record to the dietician.
The participants were informed that they should not change their dietary intake during the dietary survey.

Food Frequency Recall Food Frequency Questionnaire 
Dietary assessment was assessed with a food frequency questionnaire (FFQ) and 409 completed the FFQ.
The food frequency questionnaire is a version of a FFQ which has previously been validated (1). In the ULSAM study the FFQ has been modified and some food items have been added. The FFQ assessed each participant's usual dietary intakes during the previous year.
The first page contains questions about the meal pattern during a usual day with multiple choice answers and there are three questions about average portion sizes for potatoes/rise/pasta, meat/fish and vegetables. Four colour photos illustrate four different portion sizes.
On the following pages there are questions about frequencies of consumption of 98 food items. It is an increasing nine-level scale including never, sometimes yearly, 1-3 times per months, once a week, 2-3 times a week, 4-6 times a week, once a day, 2-3 times a day, and four or more times a day. There were no standard portions available for this age group in Sweden. Therefore we (Brita Karlström, Erika Olsson) worked up new portion sizes for this age group. The weight/size of the portion or food items were mainly taken from the Hulk study (2) and from the Swedish National Food Administration, NFA, (PC-kost 4.03).
In order to calculate energy and nutrient intake from the FFQ and not only the food frequency, a computerised program was developed by Niclas Håkansson, Department of Environmental Medicine/Nutritional Epidemiology. The frequencies of the food groups/items were linked to the data program and the four different portion sizes were also linked to different dishes and frequency. Intakes of nutrients were calculated by multiplying the frequency of consumption of each food by the nutrient content.
References 
1. Johansson I et al. Validation and calibration of food-frequency questionnaire measurements in the Northern Sweden health and Disease cohort. Public Health Nutrition 2002; 5:487-96 Public health Nutrition 2002 
2. Becker W. Befolkningens kostvanor och näringsintag. Metod och resultatanalys. Livsmedelsverket 1994. 

Questionnaire

In the investigation of the 82-year-old men, self-administered questionnaires were used. The first contained information on general and medical background, living conditions, and memory functions. The second is the "Psychological General Well-being (PGWB) INDEX" (© I.Wiklund/E.Dimenäs 1989).

Leisure Time Physical Activity (W106) was derived as follows if Q063=1 then W106=4; else if Q062=1 then W106=3; else if Q061=1 then W106=2; else if Q060=1 then W106=1;