3.1. Study subjects and design
This thesis is based on sub-studies from four study populations, the EXCADI trial (154), the CADENCE study (155), the NORSEMAN study (156) and the OMEMI trial (157). Each will be presented
separately below.
The EXCADI trial (Paper I)
The EXercise training in patients with Coronary Artery disease and type 2 DIabetes (EXCADI) study population were patients with combined T2DM and angiographically verified CAD (n=137), included in a randomized trial investigating the effect of 12 months combined resistance and endurance training on VO2max and glucose control between August 2010 and March 2012 (154). Patients were randomized 1:1 after an initial CPET, fasting blood sampling and clinical investigation, to the exercise intervention or to a control group with conventional follow-up by their general practitioner. The controls were not discouraged to exercise. The CPET and blood samples were repeated after the intervention period. 123 patients completed the study. Patients with the lowest adherence to the training intervention (< 40% adherence) were excluded (n=9) from the per protocol analyses, thus 114 patients were analysed for the intervention effect.
The CADENCE study (Paper II)
The CADENCE study included patients referred to an outpatient exercise stress test (EST) due to symptoms suggestive of CAD (n=327). Patients ≥18 years of age, both genders, with an intermediate or high pre-test prognostic risk score (Morise score ≥ 9 points) were included between December 2011 and October 2017 (158). The initial aims of the study were to investigate the effect of short-term strenuous exercise on troponin T release for the diagnosis of CAD (155). Blood samples were drawn before and within 5 min after finishing the EST. All patients underwent coronary angiography.
For the purpose of the sub-study included in this thesis, patients were grouped according to the degree of coronary artery stenosis.
41 The NORSEMAN study (Paper III)
The Norseman Xtreme Triathlon race of 2019 is a full ironman distance triathlon (3800m open water swimming, 180km bicycling and 42.2 km running), with a total climb of 5200 metres, from which volunteer participants (n=44) were recruited by email correspondence prior to the race. The
participants were all healthy athletes. Blood samples were drawn before the race, as soon as possible after the race (n=37), the day after the race (n=36) and, in a sub-set of participants available for follow-up (n=9), one week after the race.
The OMEMI trial (Paper IV)
The OMEMI trial was a prospective, randomized, placebo-controlled, double blinded multicentre trial designed to analyse the effect of a 2 year intervention with 1.8 g n-3 PUFA per day compared to placebo on cardiovascular endpoints in patients aged 72-80 years who had undergone an AMI 2-8 weeks prior to inclusion (159). Clinical examination and recordings, fasting blood samples and dietary recordings were carried out at baseline before the randomization. For the present work, baseline data from a subset of patients, all included consecutively at one site (Oslo University Hospital, Ullevål), in which subcutaneous adipose tissue (SAT) samples were available, was used (n=428).
3.2. Exercise testing
A CPET was performed on a treadmill, using a modified Balke protocol (160) in all patients before randomization, and again within one week after the intervention period (Paper I). Participants warmed up at 4% incline at an initial speed of 2.8, 3.8 or 4.8 km/hr, and after three minutes the inclination was increased every 60 sec by 2% to a maximum of 20%. If the participant was still able to keep going, the speed was increased by 0.5 km/hr until exhaustion or until ended by the physician for safety reasons (161). Ventilation, oxygen and carbon dioxide content was measured continuously breath-by-breath by a Hans Rudolph two-way breathing mask (2700 series; Hans Rudolph Inc, Kansas City, USA) connected to a metabolic cart (Vmax SensorMedics, Yorba Linda, USA). VO2peak was defined as the highest average oxygen uptake measured during consecutive 30 sec, and was also included if
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the test had to be terminated early for safety reasons or functional limitations. Time to exhaustion was registered, maximal RER measured and the AT was estimated by the ventilatory equivalent method (114).
An EST was performed on an electrical bicycle ergometer (Schiller CS-200 Excellence, Switzerland or Ergoline, Germany) (Paper II). The test was monitored by a physician and nursing staff, while a continuous 12-lead computerized ECG was recorded. Patients were instructed to maintain a
pedalling rate (cadence) of around 65 rpm. The initial work load was 30 watts (W) for women and 50 W for men, increasing gradually by 10 W per min until exhaustion unless terminated by the
physician. Reasons for actively terminating of the test were development of ECG changes such as ST-segment elevation or ST-ST-segment depression in leads without Q waves, arrhythmias increasing through exercise, chest pain, insufficient chronotropic response to exercise, and insufficient or exaggerative hypertensive response (drop in systolic blood pressure >10 mm Hg despite an increase in workload, or systolic blood pressure ≥ 250 mmHg or diastolic blood pressure ≥ 115 mmHg, respectively) (155, 162).
The Borg scale of RPE was used in both study settings to assess the participants level of exhaustion (117). The maximal RPE reached during the CPET was registered to estimate how many of the participants reached near maximal effort (RPE >17) (Paper I). During the EST (Paper II), participants were asked about their RPE every three minutes. Borg´s RPE was also used to guide the level of intensity during the group training sessions as part of the exercise intervention in Paper I.
3.3. Exercise intervention
In paper I, patients randomized to the intervention group participated in a 12 month long combined resistance and endurance training programme (154). The training programme and the trainings were planned and conducted in collaboration with the Norwegian School of Sport Sciences, Oslo, Norway.
The programme consisted of 150 minutes of training per week, divided into two group-based training sessions of about 60 min supervised by qualified instructors, and one individual home-based session
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of 30 min. Resistance training of series of 10-15 repetitions with free weights constituted approximately one third of the total training time, and the remaining two thirds consisted of endurance training including spinning on a bike, up-hill walking or running, or interval step training.
Each of the group-based training sessions included at least 5-15 min of high intensity interval training (RPE ≥15), as well as warm up and cool down. The same two instructors supervised all of the group-based training sessions, making it easier to individualize the training programme, and ensure training progression.
The home-based exercise sessions consisted of optional activities according to each participant’s interest and was registered in exercise diaries. Attendance to the group sessions was also registered, and together with the exercise diary, a total adherence to the exercise programme was calculated for each participant at the end of the 12 months (0-100%).
3.4. Coronary angiography
All study participants in the CADENCE study (Paper II) were referred to coronary angiography after completing the EST and blood sampling. Most coronary angiographies were performed using radial access. An interventional cardiologist described all coronary angiograms after performing the angiography, and a single investigator then went through all procedure descriptions, dividing the patients into three groups: (I) Significant CAD was defined as (i) ≥75% stenosis in any coronary segment, (ii) any stenosis the angiographer considered clinically relevant or decided should be treated with PCI or CABG surgery and (iii) previous PCI; (II) Non-significant CAD included patients with the range of atherosclerotic changes from minimal wall changes to <75% stenosis, and (III) No CAD was defined as no wall changes or stenotic segments. A senior investigator was consulted if the investigator was in doubt.
All included patients in the EXCADI trial had angiographically verified CAD prior to inclusion (Paper I).
All included patients in the OMEMI trial had recently undergone an AMI, verified angiographically (Paper IV).
44 3.5. Laboratory analyses
Routine blood samples, including HbA1c, CRP, insulin, C-peptide, creatinine etc., were carried out by the local hospital at the routine laboratory (Paper I, II and IV). High-sensitive cardiac Troponin T (hs-cTnT) (Paper III) was also performed by conventional methods. The lower level of blank was 3 ng/L and the lower level of detection was 5 ng/L. For statistical analyses, the level was set to 1.5 ng/L in samples below limit of blank (Paper III). Analysis of NT-proBNP was conducted by a certified clinical laboratory (Fürst medisinsk laboratorium, Oslo, Norway) (Paper III). In samples below the level of detection, the level was set to 20 ng/L.
A biobank was established in all four trials. All samples were processed after standardized protocols described in each paper, and stored at − 80 °C until analysis.
PAXgene tubes (PreAnalytix GmbH, Hombrechtikon, Switzerland) were used for the collection of RNA from circulating leukocytes for analysis of the gene expression of TLR4 (Paper II). SAT samples were taken from the gluteal region for analysis of the gene expression of CD14, LBP, TLR4 and TLR2 (Paper IV). Both PAXgene tubes and SAT samples were kept frozen at − 80 °C until RNA extraction
3.5.1. ELISA methods
Commercially available enzyme-linked immunosorbent assays (ELISAs) were used for analysis of circulating levels of sCD14 (R&D Systems Europe, Abingdon, Oxon, UK), LBP (Hycult Biotech, Uden, the Netherlands) (all four Papers) and I-FABP (Hycult Biotech, Uden, the Netherlands) (Paper I, II and III). In samples below level of I-FABP detection, the level was set to the lowest standard, 47 pg/mL.
High sensitivity CRP (hsCRP) was measured in serum by ELISA (DRG Instruments, Marburg/Lahn, Germany) (Paper IV). The inter-assay coefficients of variation (CVs) are given in each paper.
45 3.5.2. LAL assay
For the analysis of LPS in the circulation, the Kinetic Chromogenic Limulus Amebocyte Lysate (LAL) Assay (Lonza BioScience, Basel, Switzerland) was used and described in detail in the papers (Paper II, III and IV). The inter-assay CVs are given in each paper.
3.5.3. Gene expression analyses
Total RNA from PAXgene tubes was isolated by use of the PAXgene® Blood RNA Kit (PreAnalytix, Qiagen, GmBH, Hombrechtikon, Switzerland) and from SAT samples by the RNeasy Lipid Tissue Mini Kit (Qiagen, GmbH, Hilden, Germany) according to the manufacturer protocol, respectively.
Details of the gene expression analyses are given in Paper II and IV. In brief, copy DNA was made from RNA, and the analyses were performed by real time PCR using commercially available TaqMan®
assays as specified in the respective papers. mRNA levels were determined with the ∆∆CT method, using β2-microglobulin (HS99999907_m1) (Applied Biosystems by Thermo Fisher Scientific, Life Technologies Corporation, Pleasanton, CA, USA) as an endogenous control and related to a reference sample, giving relative quantification (RQ) (163).
3.5.4. Diet registration
Patients in the OMEMI trial were asked to fill out a detailed diet registration using the SmartDiet form, a validated food frequency questionnaire (164) (Paper IV). The registration was based on the patients’ recollection to 14 questions about their intake of different foods, where each of the different items were scored giving a total summary of between 15 and 45 points. The scores were further subdivided into three groups, where a score of 15-27 points was defined as an unhealthy diet (“poor”), 28-35 points was defined as intermediate (“intermediate”) and a score >35 points was defined as a heathy diet (“healthy”).
46 3.6. Statistical analyses
Throughout the four papers, demographic data are given as proportions, mean (± SD) or median (25th and 75th percentiles) depending on the data distribution. Mostly, non-parametric statistics were used. Changes in biomarkers, delta values, are given as absolute and relative changes.
Differences between two groups were analysed by Chi square test for categorical data and Mann–
Whitney U test as appropriate for continuous data. Differences between groups of three were analysed by the Kruskal-Wallis test. Friedman’s test was applied to examine for differences between more than two time points. If the Friedman’s test was significant, changes between two time points were analysed by Wilcoxon matched pairs Signed Rank test. Differences in changes and relative changes between groups were analysed by Mann–Whitney U test.
Correlation analyses were performed by Spearman’s Rho, corrected for multiple testing with Bonferroni correction where appropriate.
In Paper II, a linear regression model was used to describe the relationship between sCD14 and training background. A univariate logistic regression model was used to estimate odds ratio for peak VO2 according to levels of sCD14, and a multivariate regression analysis was used to adjust for variables known to influence peak VO2 (age, sex and BMI) (Paper I). Receiver operating characteristic (ROC) curve was performed to explore any predictive value of I-FABP for the presence of
microvascular complications (Paper I).
Statistical calculations were performed by SPSS version 25 (SPSS, Inc., Chicago, IL, USA) (Paper I), and by Stata SE version 15 (StataCorp LLC, College Station, TX, USA) (Paper II, III and IV). p-values < 0.05 were considered statistically significant throughout.
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