In the cross-sectional Lifestyle, biomarkers and atherosclerosis (LBA) study conducted at Örebro University, Sweden, young, self-reported healthy adults have been examined for early signs of atherosclerosis. Recruitment was done by advertisement at the university, in local newspapers and in social media. For more detailed information, see (13). Uppsala ethics committee approved the study design. DNR: 2014/224. All participants gave their written consent to participate, and were informed that they could terminate their participation at any time.
Body fat in percent and BMI
Height, weight and percentage of body fat, were measured with the study participants in fasting state. Height was measured to the nearest 0.5 cm with a wall mounted stadiometer. The participants were standing straight without shoes, feet together and with the arms extended along the body. Weight was measured to the nearest 0.1 kg, and percentage of body fat was calculated, using an impedance body composition analyser (Tanita BC-418 MA, Amsterdam, Netherlands). The study participants were standing barefoot on the conductive equipment, holding metal handles, according to the manufacturer’s guidelines. Adjustments were made as recommended by the instruction manual (21), with 1 kg for clothes and standard setting was used. The participant’s age, sex and length were registered in the body composition analyser. Data on body weight and body fat (%) was collected from the analyser and BMI (kg/m2) was calculated by the equipment.
Serum biomarkers
Blood samples were collected from the participants after 20 minutes of rest, following an 8-12h fasting period. The area for venepuncture was cleaned and the venepuncture was performed with a 21 gauge butterfly needle (Greiner Bio-One International GmbH, Vacuette®, Rainbach im Mühlkreis, Austria). After the blood collection, the tubes (BD Vacutainer; BD AB, Stockholm, Sweden) were gently inverted several times. Blood for analysing high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) were collected into lithium-heparin tubes, and plasma was obtained by centrifugation for 8 min at 2000xg in room temperature. A citric acid-citrate-NaF tube was used to collect blood to analyse glucose. Serum for insulin analysis was obtained by collecting blood in a standard serum tube with clot activating substances. The blood samples were allowed to clot for at least 30 min before centrifugation for 8 min at 2000xg in room temperature. All tubes were afterwards placed in +4o C until transportation and analyses at the accredited clinical chemistry laboratory at Örebro university hospital.
HDL-C, LDL-C and glucose (mmol/L) were analysed on an Ortho Clinical Diagnostics TM (Clinical Chemistry instruments, Vitros 5,1TM FS, Raritan, New Jersey, U.S.A.). The method was dry chemistry (colorimetric method) according to the manufacturer’s (Orthos) instructions. Insulin (mU/L) was analysed on an Architect instrument (i2000SR from Abbott, Illinois, U.S.A.), with their reagent according to their instructions on antibody-based technologies.
Homeostasis model assessment of insulin resistance (HOMA-IR) was calculated by using the mathematical equation by Matthews, (insulin (mU/ml)*glucose (mmol/L)/22.5). In the present study the HOMA-IR value was used as a measure of insulin resistance (22).
Mean arterial pressure (MAP) and resting heart rate (HR)
Blood pressure and heart rate were measured after approximately 10-15 minutes rest using a digital automated device (GE Healthcare, Dinamap V100, Buckinghamshire, UK) with Dura-Cuf (GE Medical Systems, GE Criticon Dura-cuf, Milkaukee, WI, US). The brachial blood pressure was measured in the left arm with the participants in a supine position. At least three measurements were done with two minutes intervals. When the difference between the latest systolic pressures were less than 5 mmHg the measurement was ended. The results for MAP and resting heart rate were reported as an average of the two latest results.
Risk for CVD - Wildman score
The Wildman risk score (23, 24) has been used to classify participants at risk for CVD. Individuals with two or more of the following characteristics were classified as being at risk according to Wildman; elevated blood pressure (130/85 mmHg), elevated triglycerides (≥1.70 mmol/L), decreased HDL-C (women < 1.30 mmol/L, men <1.04 mmol/L), elevated glucose (≥5.6 mmol/L), Insulin resistance (HOMA-IR >2.52), and elevated hs-CRP (>5.07 mg/L).
Moderate- and vigorous physical activity (MVPA)
The subject’s physical activity (PA) was measured with an accelerometer (ActiGraph, model GT3X+, Pensacola, FL, USA). The participants were instructed to wear the accelerometer on an elastic belt around their waist in the middle of their lower back during all waking time for one week. The accelerometer data was processed and analysed with the Actilife software (ActiLife, version 6.13.3, ActiGraph, Pensacola, FL, USA). The accelerometer was initialized with a sampling frequency of 30 Hz, and the vertical axis acceleration with 60-s epoch were used. Non-wear time was defined by an interval of at least 60 minutes of 0 counts per minute with an allowance for maximum 2 minutes of counts between 0-100. The participants included in the analyses needed at least 10 hours or more of wear time on the days of measurement (25). The cut-off points used to define moderate- and vigorous physical activity (MVPA) was ≥2020 counts (26). Time spent in MVPA, have been presented as minutes/day. MVPA categories were defined as follow: ≥ 30 minutes/day and < 30 minutes/day.
Cardiorespiratory fitness (CRF) measured as maximal oxygen uptake (VO2 max)
To measure CRF, a modified two point submaximal Åstrand exercise test was performed and maximal oxygen uptake (VO2 max) was calculated (13, 27). The exercise test was done on a Monark 939E (Monark Sports & Medical, Monark 939E, Vansbro, Sweden) with ECG registration to monitor heart rate (Cardiolex, EC Sense, Solna, Sweden). The exercise test started on an individually adjusted level between 50 and 100 W depending on the participants’ exercise habits. The cycling continued until the first steady-state level was reached, and then the workload was increased to reach next steady-state level. The exercise test ended when the participant reached steady state at two workload levels, with a heart rate above 130 beats/min on the first level and above 150 beats/min on the second level. The estimated VO2 max was calculated by the heart rates at the two steady-state levels and the expected oxygen consumption per work rate, by using the equation of the straight line. Maximal heart rate was estimated through the formula 220 - age in years. The study participants were categorized as having low, normal or high VO2 max according to European reference values (28). Categories for VO2 max for women were: low (≤ 30 ml/kg/min), normal (30.1 - 39.9 ml/kg/min), and high (≥ 40 ml/kg/min) VO2 max. Categories for men were: low (≤ 40 ml/kg/min), normal (40.1 – 49.9 ml/kg/min), and high (≥ 50 ml/kg/min) VO2 max.
Handgrip strength
Muscle strength was measured by Dynamometer (Fabrication Enterprices inc, Baseline® HiRes™ hydraulic hand dynamometer, Irvington. NY, US). First, the hand size was measured with a measuring tape, and the dynamometer was adjusted to fit the size of the hand (29). Handgrip strength was measured in the dominant hand. The participants sat with the arm in a 90° angle. All participants first performed one practice test, and thereafter three measurements, with one minute rest in between. The result was calculated as an average of the three measurements. The study participants were categorized as having low, normal or high muscular strength according to sex specific reference values (30). Limits for handgrip strength categories for females were: low ≤ 22 kg, normal 22.1- 34.9 kg, and high ≥ 35 kg. For males the corresponding levels were: low ≤ 37 kg, normal 37.1 – 56.9 kg, and high ≥ 57 kg.
Sleep habits
All study participants filled in a validated computerized questionnaire about their general physical and mental health (31). In the questionnaire two questions on sleep duration and experienced quality of sleep were added. The first question asked was about sleep duration and the participants were asked if they on average sleep less than 6 hours, 6 to 7 hours or more than 7 hours per night. In the second question the participants were asked, A: Do you experience good quality of sleep? B: Do you experience anxious sleep with several awakenings per night? C: Do you often have difficult to fall asleep? In the second question the participants could chose to answer yes or no.
Food habits
The participants also filled in the computerized food frequency questionnaire “Food habit choice” from the Swedish national food agency (32). The questions were based on the dietary recommendations from the Swedish national food agency (33), and the results were based on the total response to the questionnaire and presented as a score from 1 to 12 points. Participants with 1 to 4 points were considered to have unhealthy food habits. Participants having 5 to 8 points were considered to have healthy food habits with potential for improvement, and participants having 9-12 points were considered as having food habits according to the recommendations from the Swedish national food agency.
Statistics
Statistical calculations were performed using Excel 2013 for windows and IBM SPSS Statistics, version 25 for Windows (IBM Corp, Armonk, NY, USA). The Kolmogorov Smirnov and the Shapiro Wilk test were used to check all variables for normal distribution. A two-sided independent Student´s t-test was used to compare means of basic characteristics and lifestyle factors between woman and men. Equal variances were assumed. Comparison of mean between sex in the non-parametric qualitative variables (MVPA and food habits score) were analysed by Mann-Whitney U test. Pearson’s correlation coefficient (r) was used to study associations between variables.
To study relationship between variables linear regression has been used. The odds ratio (OR) of belonging to the category “At risk according to Wildman” was analysed by logistic regression, first by entering the lifestyle factors one by one and thereafter by entering all lifestyle factors in the same model. In the logistic regression analyses the variables were coded in an “increased risk” order i.e. for VO2 max, sleep duration, food habits, and handgrip strength (at the levels high/normal/low), and for MVPA (at the levels high/low). The probability to be at risk according to Wildman risk score, was expressed as exponential beta, Exp (B). The levels of significance have been expressed as P<0.05=*, P<0.01=** or P<0.001=***. To illustrate correlations and quartiles Excel for windows have been used.