The following methodological discussion was inspired by the STROBE (STrengthening the Reporting of OBservational studies in Epidemiology) initiative (195).
5.1.1 Study design
The design of the Vitality study was cross-sectional with a longitudinal component, e.g., related to psychometrical testing of questionnaires. In the current thesis, merely cross-sectional data were used for analyses and interpretation. A clear limitation of cross-cross-sectional studies is that conclusions on causality cannot be clearly drawn. Consequently, it is beyond the scope of this thesis to conclude on the directional relationship between the various endpoints themselves, and some of the factors significantly associated with these endpoints.
For instance, the cross-sectional design hampers our ability to conclude on a clear directional relationship between vitamin D deficiency and disease activity in IBD,
5.1.2 Recruitment and selection bias
In the Vitality study, patients were included at nine different hospitals in the southeastern and western part of Norway. The centers were both university hospitals and community hospitals.
This might cause heterogeneity in the recruited population. However, Norway has a high prevalence of IBD patients, and all hospitals have a wide experience in treating these patients.
The guidelines for treatment and follow-up of patients with IBD were also quite similar across the centers, as they are based on national and European guidelines and consensus. Thus, large variations between the populations were not expected. However, we did find that a higher proportion of those included at university hospitals were treated with biologicals than those in the community hospitals. Moreover, we did not formally register patients who either declined to participate in the study or could have been invited to participate. Consequently, we cannot completely rule out the risk of selection bias.
5.1.3 Data collection and seasonal variation
The inclusion period in the Vitality study lasted one year. This means that some patients had their data collected during winter and some during summer. When looking at the distribution
of vitamin D measurements specifically, we found, that the number of measurements were evenly distributed between the summer and winter months. Moreover, no seasonal variation was found in vitamin D levels, which may be due to the general recommendations of taking supplements during winter. Even though patients were included at centers situated at different geographical locations, all were from the southeastern and western parts of Norway, and none from northern Norway. We believe that the variation in sun exposure in southern Norway is negligible, and that the differences between individual patients may be more important. A limitation, nevertheless, is that sun exposure was not investigated. The reported intake of fish and other foods rich in vitamin D was small, as reported in Paper I, and is unlikely to have influenced the results.
5.1.4 Sample size
Since the Vitality study was an explorative, observational study without testing of any specific hypothesis, a formal power analysis was not feasible. The study intended to
investigate the prevalence of vitamin D deficiency as well as other outcomes, and to generate hypotheses from possible associations to the main study outcomes. During the planning of the study, however, the inclusion target was set at n = 400, which was considered feasible during the inclusion period of one year. This target was reached.
5.1.5 Completeness of data
We consider the completeness of data a strength in our study. Only a few patients had missing vitamin D analyses, and even though missing data might be expected when using PROMs, the numbers of missing data from questionnaires in the current study was very low.
5.1.6 Data sources and measurements
During the planning phase of the vitality study, the methods of vitamin D measurement was thoroughly discussed in the research team as well as with external experts in the field. The method of Vitamin D measurement may be of importance when comparing studies of potential effects and associations with vitamin D. The two most common methods,
immunoassays and LC-MS/MS, both measure the total amount of vitamin D metabolites 25-OH-D2 and 25-OH-D3 in serum. Some immunoassays do not identify all 25-25-OH-D2 and may produce lower results than the actual value. The LC-MS/MS method is therefore
recommended. Following recommendations and discussions, the latter method was chosen in
the current study, which we indeed believe strengthens the validity of our results. When comparing results from different studies, and translating research data into recommendations for clinical practice, which method used needs to be considered.
In order to strengthen the validity of our results and interpretations furthermore, the research team chose to perform the analysis of serum 25-OH-D for all patients at the Hormone
laboratory (Department of Medical Biochemistry, Oslo University Hospital, Norway). At the hormone laboratory, an in-house developed LC-MS/MS method that has been compared to the LC-MS/MS method used in VDSP, with good compliance, was used (51). The laboratory participated in the Vitamin D External Quality Assessment Scheme (DEQAS) for total 25-OH-D. Furthermore, the laboratory is accredited by the Norwegian Accreditation as a testing laboratory and complies with the requirements of NS-EN ISO/IEC 17025. This was done to reduce potential variations in measurements at local hospital laboratories that otherwise could have affected the results.
Vitamin D deficiency was defined as 25-OH-D concentration < 50 nmol/L, and vitamin D insufficiency was defined as a 25-OH-D concentration of 50-75 nmol/L. If we had conducted our analyses with a cut-off for vitamin D deficiency at < 75 nmol/L, this would have been less meaningful as this target was not established at the time. Additionally, relatively few patients had sufficient levels of 25-OH-D.
Besides vitamin D, all other biochemical analyses were performed at the local laboratories at each hospital. The measurement of C-reactive protein (CRP) is standardized between the different laboratories, and only smaller variations may be expected. In the study we chose a CRP level of 5 mg/L or higher to indicate active inflammation as recommended in previous reports (181, 182). There may however be a lack of association between systemic
inflammation, as measured by CRP, and intestinal inflammation, as many patients with IBD have normal CRP levels even in active disease (196).
Fecal calprotectin, however, is well correlated with intestinal inflammation in UC, but in CD inflammation may be present even with lower levels of fecal calprotectin, especially in
localized, small-bowel disease (183). Fecal calprotectin < 100 mg/kg was set as the cut-off for disease in remission, while higher levels were defined as active inflammation. This was the most relevant for the method used to analyze fecal calprotectin (183). Additionally, more than
half of patients with CD had colonic involvement, and thus the cut-off was deemed relevant in evaluating intestinal inflammation also in CD.
Clinical disease activity was measured using disease activity scores. Symptoms, however, do not always represent active inflammation, and clinical indices may therefore not always correspond to objective assessments of disease activity. Associations of different outcomes to elevated symptom scores therefore need to be interpreted with some caution.
5.1.7 Use of questionnaires
Paper-based questionnaires were used to investigate fatigue, pain severity, sleep disturbance and psychological symptoms. The choice of instrument is highly relevant in the design of a study. When comparing results from different studies, the questionnaire used to measure for example fatigue, is important, since different questionnaires measure different aspects of fatigue. In addition, there may be differences both in the number of cases and severity reported with the different instruments. Our rationale for choosing the FQ to evaluate fatigue in the current study and thesis was based on its prior validation and testing in the general Norwegian population and IBD, as well as the fact that multidimensional fatigue scales are recommended (114, 117, 122, 123). The multidimensionality of the FQ allowed us to evaluate both physical- and mental fatigue with vitamin D status.
The questionnaire used for measuring pain severity was the BPI, which also have been translated into Norwegian and validated in IBD patients (190, 193). Questionnaires validated in the population of interest, such as this, are less likely to be subject to measurement errors.
To be able to adjust for the potential influence of psychological factors in the experience of fatigue and pain severity, the HADS questionnaire was used to measure depressive symptoms and anxiety symptoms. Likewise, sleep disturbance was assessed to adjust for this factor in the analyses of fatigue.
As discussed earlier, self-reporting of symptoms may constitute a source of bias, meaning the deviation between the reported and true values of the same measure. Recall bias may be important when reporting symptoms back in time such as chronic fatigue or clinical outcomes such as relapses. The patients were asked how many relapses they had during the previous year, and the accuracy and definition of reported relapses were not verified by other data
sources such as medical records. On the other hand, patients with chronic conditions like IBD tend to keep a good record of their relapses. Cases of fatigue were identified using the
conventional cut-off of dichotomized FQ scores ≥ 4 with a duration ≥ 6 months (6, 117). The patients were asked to recall symptoms of fatigue over the last four weeks, and if they
reported fatigue, for how long they had the symptoms. The FQ, however, has been validated in IBD patients, and prevalence of fatigue in our study was comparable to other studies in IBD (6, 114).
Response shift is also of importance in self-reporting of symptoms in chronic disease.
Response shift may be especially relevant to consider in self-reporting of symptoms like fatigue and pain, as patients may become used to living with them over time. As mentioned earlier, patient support and coping mechanisms aimed at helping the patient adapt to living with chronic disease, may lead to a positive response shift that has been encouraged.
In relation to this, the duration of disease was longer in CD patients, as compared to UC patients in the study. It may be speculated that this can lead to under-reporting in the CD group resulting in potential measurement errors, but this remains a theoretical concern not supported by our results.
5.1.8 Statistical methods
All statistical analyses were supervised by an experienced biostatistician. Parametric and non-parametric tests were used where appropriate. Different types of regression analyses were fitted to investigate the various outcomes as needed, including univariate and multivariate analyses using both logistic regression and ordinal regression. All analyses were done
separately between UC and CD, as this was considered relevant form a clinical point of view as outlined in the aims of the study.
5.1.9 Funding and editorial support
The study was supported by a research grant from Tillotts Pharma and Østfold Hospital Trust, Norway. English language editing was provided by American Journal Experts for paper I and paper II. Editorial support was provided by Cambridge Medical Ltd in agreement with
Pharmacosmos AS, Denmark for paper III. The sponsors had no role in the design, execution, interpretation of the study, or writing of the papers.
5.2 Discussion of main findings