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6. Discussion

6.1. Methodological considerations

6.1.1. Study populations and design

In general, none of the studies that are the basis for this thesis were designed to evaluate gut leakage markers in particular, and power calculations were not performed for these sub studies. Information on some important factors known to potentially influence gut leakage markers may therefore be missing, which are i) the usage of antibiotics, ii) the usage of drugs with anti-inflammatory properties and iii) the participants specific diet, as stated in the respective papers. This will be discussed

thoroughly.

Paper I is based on a randomized, controlled trial designed to evaluate the effect of a 1 year exercise intervention on glycaemic control in patients with CAD and T2DM. Patients with ongoing infections were excluded, which is a strength when considering gut leakage markers and activation of

immunological pathways. Secondly, as it is a randomized trial, we must assume that any other factors influencing the gut leakage markers in these patients are evenly distributed between the two

intervention groups. However, due to the nature of exercise training, patients were not blinded as to which group they were in. It is known that a healthier diet tends to accompany adherence to an exercise programme, whether intentionally or unintentionally from the participant´s side. On the other hand, we also experienced some patients being unsatisfied with being randomized to the control group as they were eager to initiate a lifestyle change and wanted to start exercising. It is therefore not unlikely that some of the patients in the control group also increased their level of physical activity or changed their diet during the 1 year intervention period, as they for ethical reasons were not discouraged to exercise (vide supra). This might have led to an underestimation of the exercise effect.

In addition to being a randomized study, a major strength of Paper I is the inclusion of patients with both CAD and T2DM. There is a lack of exercise intervention trials in patients with both diseases and

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with longstanding disease. It was a group of patients quite heavily burdened with comorbidities, as the exclusion criteria were mainly related to serious illnesses associated with life expectancy under 1 year, and other conditions incompatible with adhering to an exercise programme. It´s generalisability thus embrace a comorbid group of patients of which we knew little about gut leakage. For the same reasons, it is difficult to transfer any conclusions from this study to other patient groups, and the results must therefore be interpreted with caution as to which patient group it represents.

The results in Paper II are based on a cohort of patients with symptoms suggestive of CAD and an intermediate or high pre-test prognostic risk score who were referred to an outpatient EST. As mentioned, it was designed to investigate cardiac biomarkers as an addition to the EST to increase the non-invasive diagnostic accuracy of significant CAD. All blood samples were therefore taken at approximately the same time both before and after the EST in all patients, which is a strength when investigating acute changes in gut leakage markers. Another strength of this study is the

generalisability to the common population in which we might expect symptomatic CAD, both regarding age and sex. Lastly, the study was performed under very well-controlled circumstances, supervised by both a physician and a nurse, continuously monitoring vital measures.

Paper III is based on a cohort of healthy athletes participating in the Norseman Xtreme Triathlon race, which was designed to investigate common biomarkers used in the clinical setting to aid in the evaluation of what is normal and what might represent pathology after such an extreme physical exertion. The results from the study may only apply to healthy, endurance trained individuals, and may not be representative for older individuals or individuals with pre-existing CAD or other cardiometabolic conditions. There was also quite a low number of females, which might influence the generalisability to both sexes. However, when investigating the females separately, all gut leakage and cardiac biomarkers followed the same pattern, although reaching lower peak values, which is in line with the current literature (166) (data not published). Lastly, one can speculate whether the results are transferable to other modes and duration of exercise. Especially the last part

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of the triathlon, the running, has been proposed to inflict a greater mechanical stress on the intestines due to the pattern of movement than does other long-lasting races such as cycling only (167). This might have influenced the levels of gut leakage markers, and the results might therefore not be transferable to other endurance sports. However, others have found intensity rather than mode of exercise to be the most important factor for gut leakage (168).

The results in paper IV are based on a sub-study of the first consecutively included patients from OUH in which SAT samples were available, all part of a larger randomized, placebo-controlled trial designed to evaluate the effects of n-3 PUFAs in elderly MI patient. However, the design of the current investigation was cross-sectional to assess associations. It is therefore not suitable for making any inferences about causality. A major strength of this study is the large number of adipose tissue samples and the detailed dietary registration.

6.1.2. Measures of circulating gut leakage markers

LPS is the only direct marker of gut leakage, as it originates from the surface of bacteria. However, not all LPS in the blood stream is necessarily from the gastrointestinal tract. LPS has been shown to translocate into the blood stream from bacteria residing in the respiratory tract, and might also originate from other sites of infection or a broken skin barrier. Nonetheless, the intestines harbours by far the most comprehensive amount of LPS, and it is therefore most likely that a detected rise in LPS to great extent represents leakage from the intestinal residence. Additionally, LPS only

represents the pool of gram negative bacteria, and does not reflect any gram positive bacterial products that can leak simultaneously.

Interpretation of LPS levels in the circulation must therefore be done with caution. As mentioned, it has a very short half-life of only a few minutes (74). This ultimately means that it represents only the current LPS-leakage, and does not reflect any translocation of LPS prior to sampling or chronic exposure. Once in the circulation, it is rapidly bound to other neutralising components of the plasma, mostly LBP, high-density lipoprotein (HDL) or other lipoproteins (169). Such sequestration makes the

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LPS undetectable to the LAL assay, and no pre-treatment of serum has so far proven successful to promote its release once it has been sequestered (72). In individuals with a high capacity for LPS clearance, levels of LPS might therefore be underestimated. Furthermore, LPS is rapidly transported to the liver for detoxification. Detoxification means removal of one or more acyl groups, thus altering the immunostimulatory potential of the LPS, which can ultimately influence its ability to activate the LAL assay (170). Lastly, the method for LPS-detection itself has many limitations, and has been criticised (72). However, we have chosen to include levels of LPS in the last three papers, as it might add valuable information, at least when interpreted with caution and together with the other gut leakage markers. Because of its volatility, it is especially valuable in the papers evaluating the acute effect of training (Paper II, Paper III), but might be of poorer value when evaluating any chronic leakage.

Both sCD14 and LBP are important pieces in the LPS-induced inflammatory pathway. However, they can both be considered acute phase proteins (78, 85), and thus represent a more general

inflammatory activation. Among other known stimuli are Gram-positive cell wall components and heat shock proteins (171). A rise in sCD14 and LBP is therefore not necessarily restricted to the burden of LPS in the circulation, and this must be taken into account when interpreting the results.

Nonetheless, they are acknowledged markers for LPS exposure, and they are also considered to be a lot more robust in the circulation than LPS, rendering them more reliable markers of chronic LPS-exposure than LPS itself (79). In addition, the ELISA methods used to detect both sCD14 and LBP are reliable.

In our work, we have not considered I-FABP as a marker of LPS-leakage, but rather a marker of injury to the intestinal endothelial barrier (102). It thus gives information on the mode of leakage, or at least indicate whether there is damage to the intestinal barrier. As it is found in the circulation in healthy individuals to a very limited degree, any rise detected will be sufficient to suggest barrier injury. Considering the single epithelial cell layer of the intestines, it is likely that leakage of LPS and

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other bacterial products accompany any injury to this layer. An absence of change or rise in I-FABP will at the same time not exclude other modes of gut leakage, such as para- or transcellular leakage.

FABPs in general have a half-life of 11 min in the circulation (106), making it an excellent marker for acute and rapid changes, such as in Papers II and III.

6.1.3. Exercise testing and intervention

All patients in Paper I performed a CPET prior to and after the intervention period, allowing us to evaluate any change in CRF after the intervention, and thus the effect of the exercise intervention. A CPET is standardised, and the measures are therefore comparable to that of other studies, as long as the participants have achieved a sufficient level of effort during the test. The risk vs. benefit of such a close-to-maximal exertion in these patients is discussed under the “ethics” section (vide supra). We observed no effect of the 1 year exercise intervention on physical fitness assessed by VO2peak. The exercise programme was designed to affect HbA1c in the best possible way, and might therefore not be the best to increase VO2peak, which is the parameter in physical fitness that has been shown to associate to levels of gut leakage markers. In addition, the adherence to exercise was somewhat low.

In general, the participants in the intervention group suffered quite a few musculoskeletal injuries and issues throughout the year, some encountered difficulties with the motivation to sustain training and some had intercurrent illnesses or events related to their disease. In the analyses we excluded patients with less than 40% adherence to the training programme to try to assess the effect of actually exercising. It may be argued that this is a somewhat low adherence cut-off, however, we believe that these numbers reflect the difficulties encountered when trying to exercise such a comorbid group of patients, and therefore probably is close to what one would achieve in clinical practice. The results are, however, not applicable to other less comorbid groups of patients or patients with other illnesses. Others have previously found gut leakage markers to improve after an exercise intervention in diabetic subjects without comorbidity followed for a much shorter period (152). Thus, also the long-standing program may have influenced the degree of adherence.

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In Paper II all patients performed an EST under close supervision. The EST was done on a bicycle ergometer, which is advantageous with the simultaneous ECG recording and measurement of vitals, as it allows for much less upper body movement than that of running or walking. This also allowed us to take the blood samples immediately after the test was completed, while the patients were still seated on the bicycle. An additional blood sample taken somewhat later would have added to our results. It is also easier to standardise the work load on a bike, as it is not a full body weight bearing exercise. Nevertheless, all participants were exercised to exhaustion. The huge drawback with an EST as compared to a CPET, is the lack of measurements of CRF such as VO2peak and the estimation of AT.

In Paper III, all individuals participated in the same race, rendering the participants exposed to the same work load, although allowing for individual regulation of the intensity. However, as this was a race, we assume that all participants came close to maximal effort during the course. A limitation to this study is the lack of formal testing of CRF. We have tried to draw conclusions between training habits and biomarkers, however, a higher training volume may not always equal a higher CRF, as physical fitness is a product of the intricate and person specific balance between correct training stimulus, restitution, diet and rest. Hours of exercise may therefore not be regarded as a substitute measurement for CRF in any way, but rather as a total time of exposure to a work load higher than that of resting.

6.1.4. Diagnosis of CAD

In Paper I, all patients were investigated with coronary angiography and diagnosed with CAD as part of clinical practise. Also in Paper IV, all participants were subject to coronary angiography prior to inclusion as part of the diagnosis of AMI. The participants in the CADENCE study (Paper II) all

underwent coronary angiography after the EST to assess degree of coronary artery stenosis. This is a strength in all three studies, as invasive coronary angiography is considered the golden standard for the diagnosis of CAD.

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In Paper II, the patients were divided into three groups according to the degree of coronary artery stenosis, based on the description of the coronary angiogram. The development of CAD is a continuous process, and it is therefore important to be aware the possible inappropriateness of dividing the patients into such theoretical subgroups. It has also been shown that the stenosis severity does not equal plaque instability, and does not necessarily predict an acute event, as less severe stenoses (<70% stenosis) have been shown to account for more cases of plaque rupture and thrombosis than severe stenoses (>70% stenosis) (172).

6.1.5. Diet and dietary registration

Information about the participants’ general diet is valuable as diet composition, and especially high fat meals might promote the translocation of LPS across the gut barrier (71). The detailed dietary registration in Paper IV is a strength of this study, although the registration was based on the patients’ recollection of their food intake. Registry based on memory might be inaccurate and can lead to both an over- or an underestimation of total calories consumed and the specific amount of the specific macro nutrients. To overcome this, the ideal is to weigh and measure all items of the participants’ diet to complete a registration, which is both time consuming and quite comprehensive.

We have therefore chosen the SmartDiet form, which is a validated scoring system, in Paper IV.

A major limitation in Paper III is the lack of controlling for the participants diets, both before, during and after the race. As it indeed was a race, it was not possible to ask the participants to show up fasting to blood sampling, and due to the median duration of 14 h 33 min (13 h 42 min, 15 h 29 min), participants were allowed to eat and drink at need during the race. This might have influenced our results, especially the concentration of LPS, which is sensitive to simultaneous or recent food intake (vide supra).

In Papers I, II and IV, the patients were fasting upon all blood sampling, thus well standardized. In Paper II, they were not allowed to eat or drink during the EST, a strength when considering acute changes in gut leakage markers considering the diets influence on especially LPS. In Paper I, the

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participants were encouraged not to change their diet during the intervention. An estimation of total caloric intake at baseline and at the end of the 1 year intervention period were made based on a 24-h dietary recall form, but no details on the diets composition was registered. The potential bias with a recall-based diet registry and the possible unintentional alteration of the diet that might accompany a lifestyle intervention have previously been addressed. Nonetheless, we observed no significant difference between the groups at baseline or after the intervention in mean energy intake (all p>0.05). We therefore chose not to include this parameter in the analysis, and have listed this as a limitation in Paper I.

6.1.6. Other factors possibly influencing the gut leakage markers

Several other factors are known to influence the gut microbiota composition, health or gut barrier integrity. We have, as far as possible, included information on drugs known to influence the gut microbiota or the intestinal barrier. In Paper III, information on the use of medication was

unavailable. However, we assume that usage of any medication amongst these healthy athletes with no previous disease is limited, and in any case mostly revolve around non-steroidal

anti-inflammatory drugs (NSAIDs).

The most important drug class with the potential to influence the microbial composition of the gut is antibiotics (173). Although most modulatory effects of antibiotics are transient, some changes have been shown to persist for years before the microbial composition fully recovers (173). Others have chosen to exclude patients on antibiotic treatment for up to 6 months prior to inclusion (141). In Papers I, II, and IV we lack information on antibiotic usage in the time prior to inclusion, but no patients were on antibiotic treatments at the time of inclusion. Also, in Paper I, any use of antibiotics during the 1 year intervention period was not recorded. However, we find it reasonable to assume that any use of antibiotics was evenly distributed between the randomized groups.

Another important class of drugs which may influence our results are statins (174). Statins are HMG-CoA-reductase inhibitors which have LDL-lowering effects, and are widely used lipid lowering agents

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in populations with cardiometabolic disease. They hold the possibility to change both the microbial composition, and perhaps more importantly the functional capacity, although the full interaction with the gut microbiota is far from established (174, 175). Additionally, they have anti-inflammatory properties which can complicate the interpretation of the gut-related inflammatory pathway we study (176). In general, drugs with anti-inflammatory effects hold the potential to mask or conceal changes in the LPS-inflammatory pathway and the low-grade chronic inflammation associated with it.

Both antiplatelet drugs (177), diabetic drugs like metformin (178) and of course

anti-inflammatory drugs such as NSAIDs and prednisolone (179) all have immune modulating properties.

NSAIDs additionally holds the potential to damage the gastrointestinal tract directly, and thus

increase gut leakage (180, 181). We have discussed these important aspects in the respective papers.

Lastly, also alcohol intake is known to affect the gut microbiota and the gut barrier (182, 183). One study has shown systemic LPS bioactivity assessed by sCD14 levels to be elevated up to 48 hours after alcohol consumption (184). Patients were not, however, asked to refrain from alcohol prior to blood sampling, and all papers lack information on alcohol intake.

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