30 May 2023: Clinical Research
Association Between Serum Vitamin D Levels and Physical Outcomes of Patients Who Underwent Rehabilitation Following Ischemic StrokeWojciech Borowicz 1ABCDEF*, Kuba Ptaszkowski 2ACDEF, Lucyna Ptaszkowska3ADEF, Joanna Rosińczuk 4ADE, Eugenia Murawska-Ciałowicz5ABCDEFG
Med Sci Monit 2023; 29:e940115
BACKGROUND: Ischemic stroke is the most common cause of disability in adults. Deficiency of vitamin D in patients with cardiovascular diseases is increasing. Only a few studies have assessed the relationship between serum vitamin D levels and functional capacity and degree of disability. This study aimed to evaluate the association between serum vitamin D levels and physical outcomes of 94 patients who underwent physical rehabilitation following ischemic stroke.
MATERIAL AND METHODS: A group of 94 patients was enrolled; however, 80 patients (61.8±6.9 years) were included. They underwent a 6-week rehabilitation using proprioceptive neuromuscular facilitation (PNF, 60 min daily), mirror therapy (MT, 30 min daily), and occupational therapy (OT, 45 min daily). The Barthel Index (BI) and modified Rankin scale (mRS) were used for functional assessments. Laboratory blood tests for serum vitamin D and insulin-like growth factor 1 (IGF-1) levels were conducted.
RESULTS: There was a significant increase in BI scores (median difference=2.0 points [pts]; P<0.001) and IGF levels (median difference=124.6 ng/ml; P<0.001) after rehabilitation. There was a significant decrease in mRS scores (median difference=7.0 pts; P<0.001), but there was no significant difference in vitamin D levels (P=0.40). The effect of age (B=-0.01, P=0.04) and serum vitamin D level (B=-0.02, P=0.01) on the BI score was demonstrated. The effect of body mass index (BMI) results (B=-0.07, P=0.02) on the mRS score was observed.
CONCLUSIONS: Lower serum vitamin D levels and more advanced age may be associated with worse functional outcomes in first-ever ischemic stroke patients.
Keywords: 25-hydroxyvitamin D, functional status, ischemic stroke, neurological rehabilitation
Ischemic stroke causes irreversible brain damage and affects people of all ages on all continents. It is the second leading cause of death , and it is the leading cause of disability in people aged over 45 years . Despite significant improvements in primary prevention, stroke continues to affect more than 1.1 million Europeans  and Chinese  each year, which indicates the severity of the problem.
Established risk factors for ischemic stroke do not explain the occurrence of the disease in one-third of patients , warranting further exploration of potential risk factors. Serum vitamin D levels are positively associated with improved cardiovascular health, particularly with a reduced risk of stroke . Recently, there has been an increased prevalence of vitamin D deficiency in patients with cardiovascular diseases, including post-stroke patients .
Vitamin D deficiency is described as a co-morbid condition in many diseases such as acute ischemic stroke, neurodevelopmental disorders and intellectual disability, and cardiovascular risk, and it is associated with increased mortality [8,9]. The discovery of the vitamin D receptor (VDR) made it possible to learn more about its effects on extraskeletal organs . The identification of calcitriol (the active form of vitamin D) receptors in various tissues of the human body confirmed the pleiotropic, multidirectional effects of vitamin D .
Zhou et al , in their systematic review and meta-analysis, confirmed the hypothesis that low vitamin D levels are associated with an increased risk of ischemic stroke, which indicates they are a possible risk factor for stroke. In contrast, higher vitamin D concentrations significantly protect against stroke.
Vitamin D is a well-known neurosteroid hormone that plays an important role in modulating cognitive processes and regulating neurotrophic signaling  for neuroprotection, neuromodulation, vascular risk factors for stroke [14–16], and inflammation [17,18]. VitaminD, both from food and that synthesized in the skin, undergoes transformations that result in the formation of 2 metabolites. Hydroxylation to 25-hydroxyvitamin D (25(OH)D), a substrate for the synthesis of the biologically active 1,25 dihydroxyvitamin D (1,25(OH)2D), occurs in liver cells, which takes place in the proximal cells of the kidneys. Both metabolites enter the bloodstream. Although 1,25(OH)2 is a biologically active form of vitamin D, its serum levels do not correlate with the state of the body’s actual supply. A marker of vitamin D content is the measurement of 25(OH)D. The half-life of the circulating metabolite in the blood is approximately 21 days and is the best indicator of the total vitamin D content in the body. In contrast, the 1,25(OH)2 present in serum has a short half-life of approximately 4 hours and determination of its content is not used for assessing vitamin D deficiency.
Many studies in the literature have assessed the relationship between vitamin D in terms of stroke risk [6,19]. Few studies have assessed the relationship between serum vitamin D levels and the functional capacity of patients and the associated degree of disability. Therefore, there was an attempt to assess the relationship between vitamin D levels in patients admitted to rehabilitation and functional outcomes. Therefore, this study aimed to evaluate the association between serum vitamin D levels and physical outcomes of 94 patients who underwent physical rehabilitation following ischemic stroke.
Material and Methods
The study was approved by the Ethics Committee of Wrocław Medical University (permission no. KB-813/2020). The study was conducted in accordance with the Good Clinical Practice Guidelines and the Declaration of Helsinki. Each person was briefed and instructed on the conduct of the experiment before signing the consent to participate. All study participants were informed of the possibility of discontinuing their participation in the project in the case of health problems or for other reasons.
The study was conducted as part of the project “Effectiveness of various therapeutic forms and their influence on nervous, muscular and vascular plasticity in patients after ischemic stroke” in collaboration with Dr. Sílvia Rocha-Rodrigues (Research Centre in Physical Activity and Health, Faculty of Sport, University of Porto, Portugal). The project was registered in the International Standard Randomized Controlled Trial Number Registry Platform (registry no. ISRCTN16891871).
A group of 94 post-ischemic stroke patients undergoing rehabilitation at the Neurological Rehabilitation Unit of the Wrocław Regional Specialist Hospital were enrolled in the study. Patients were qualified to participate in the research by a team consisting of a physician-specialist in medical rehabilitation, a neurologist, a neurologopedist, a clinical psychologist, and a physiotherapist. Inclusion criteria for the study included first-ever ischemic stroke, time of onset 2 weeks before admission for early neurological rehabilitation, stroke confirmed by magnetic resonance imaging (MRI) or computed tomography (CT), age >18 years, no contraindications to participate in the experiment (consent of attending physician), no neurological comorbidities, and written informed consent of the patient to participate in the study. Exclusion criteria included patients with an infection in the past 2 weeks, patients who took vitamin D and its derivatives or calcium in the past 3 months, patients with liver and kidney disorders, patients with thyroid disorders, patients with aphasia, and patients who did not provide consent. A group of 80 patients met the criterion of participating in 2 follow-up examinations was finally qualified for the study. There were 25 women (31.3%) and 55 men (68.7%) in that group. Patients were provided with proprioceptive neuromuscular facilitation (PNF) and mirror therapy (MT) for 42 days. At 6-week intervals, patients had blood drawn for routine tests and selected biochemical parameters were determined in the remaining blood. All tests and surveys were carried out by the same physician.
On the day of admission, all patients underwent standard neurological examination, basic anthropometric measurements, and stroke severity assessment using the National Institutes of Health Stroke Scale (NIHSS). In terms of primary outcomes, functional tests were performed using Barthel Index (BI) and modified Rankin scale (mRS).
The BI is a commonly used scale to assess the level of functional independence in patients undergoing physiotherapy after stroke. The BI measures deficits in self-care, mobility, and sphincter control. Scores ranging from 0 to 20 points are interpreted as follows: ≤4 points – very severe disability; 5–9 points – severe disability; 10–14 points – moderate severity of disability; 15–19 points – mild disability; 20 points – full independence.
MODIFIED RANKIN SCALE:
The mRS is a commonly used tool to assess functional outcome after a stroke. It assigns a score ranging from 0 (no symptoms) to 6 (death), with higher scores indicating more severe disability. The scale is often used to assess a patient’s ability to carry out activities of daily living, such as eating, dressing, and mobility. The mRS has been shown to have good inter-rater reliability and validity, and is widely used in both clinical practice and stroke research.
LABORATORY BLOOD TESTS:
Patients had their blood routinely drawn from the ulnar vein for basic laboratory tests such as complete blood count (CBC) and urinalysis. The following concentrations were determined: vitamin D and IGF-1 levels. The serum levels of those substances were determined on admission to the unit and after 6 weeks of rehabilitation training. Each patient had blood drawn from an ulnar vein, always at the same time, at 6: 30 a.m., on an empty stomach. Laboratory tests were performed at the Scientific Laboratory of the Research and Development Center at the Regional Specialized Hospital in Wrocław. Performing biochemical tests at the same time, using reagents from a single production batch, and ensuring the same measurement conditions was intended to minimize the occurrence of external, random errors that could alter the results of biochemical analyses. The tests were performed in the laboratory on an Alinity ci analyzer using reagents from Abbott (Chicago, Illinois, USA). This is a widely used, standardized immunochemical assay using a chemiluminescent microparticle immunoassay (CMIA) used to quantify 25(OH)D in human serum and plasma on an Alinity ci analyzer. The Alinity ci 25-OH Vitamin D test has been standardized against the National Institute of Standards & Technology Standard Reference Material 2972 (NIST SRM 2972). The test is used as an aid in assessing adequacy of vitamin D levels. It is a one-step immunochemical test with delayed conjugate addition, dedicated to the quantitative determination of 25-hydroxyvitamin D in human serum and plasma using microparticles and a chemiluminescent marker (CMIA). Paramagnetic microparticles coated with anti-vitamin D antibodies and assay diluent are added to the test sample, and the whole mixture was incubated. The 25-hydroxyvitamin D present in the sample was removed from the vitamin D-binding protein and then binds to the anti-vitamin D antibodies, coating the microparticles. A conjugate containing acridine-labeled vitamin D was added to form the reaction mixture. The reaction mixture was incubated. After a wash cycle, the Pre-Trigger Solution and the Trigger Solution that triggers the Trigger Solution reaction were added. The intensity of the signal produced by the chemiluminescence reaction was measured in relative light units (RLU), based on the relationship between the amount of 25-hydroxyvitamin D in the sample and the RLU values measured by the optical system. A normal serum vitamin D level was defined as a 25(OH)D concentration >30 ng/ml. Vitamin D deficiency was defined as a concentration of 25(OH)D <30 ng/ml.
Once stabilized during the early neurological rehabilitation period, post-ischemic stroke patients may develop a wide variety of disorders, the clinical picture of which will depend on the nature and extent of damaged brain tissues. Movement disorders are one of the most common clinical manifestations. Rehabilitation treatment (rehabilitation training) focuses on regaining motor function to a degree that enables patients to achieve and meet their self-care needs. Patients selected for the project were rehabilitated with neurophysiological methods (PNF and MT) for 5 days a week. Rehabilitation using the PNF method was oriented towards improvement of a specific lost function, not just a structure. The overriding aim of the therapy was to help the patient achieve the highest possible functional level. Each unit of treatment for mobility issues lasted 60 minutes. Moreover, each patient additionally had MT applied daily for 30 minutes. The rehabilitation training was 6 weeks long. Also, each patient had daily one-to-one sessions with a clinical psychologist for 30 minutes and an additional 45 minutes of occupational therapy (OT). For patient safety reasons, blood pressure and heart rate were measured before each kinesitherapy unit. Since patients with diabetes were also included in the study group, electroencephalography (ECG) changes indicative of myocardial hypoxia, which may also occur during exercise, were an absolute precondition for discontinuing the rehabilitation training. The rehabilitation training has always been individually tailored to each patient and their current abilities and needs, taking into account the patient’s capabilities.
The sample size analysis was based on the main objective of the study, which was to assess changes in vitamin D levels before and after 6 weeks of rehabilitation. Before the start of the study, it was assumed that there would be an increase in vitamin D levels. The minimum sample size needed to detect a difference of 20% assuming alpha=5%, power=80% and confidence level=95% is 79 patients. The sample size analysis was performed using the G*Power software. Statistical power analyses were performed using G*Power 3.1
Statistical analysis was performed using Statistica 13 software (TIBCO, Inc., Palo Alto, USA). Arithmetic means and standard deviations or medians, upper and lower quartiles and the range of variability (minimum and maximum value) were calculated for measurable variables. Prevalence (%) was calculated for qualitative variables. All studied quantitative variables were verified using the Shapiro-Wilk test to determine distribution type. Comparisons between pre- and post-rehabilitation scores were made using non-parametric Wilcoxon test. The level of α=0.05 was used for all comparisons. Moreover, the analysis of the impact of selected variables on activities of daily living (BI) and disability/dependence in the daily activities (mRS) was performed using a linear regression (univariate model). The unstandardized and standardized regression coefficients, standard error and level of statistical significance were determined. The next step was to develop a multivariate model (stepwise progressive method), taking into account variables whose
The study ultimately included 80 patients meeting the participation criterion; the mean age of participants was 61.8±6.9. In this group, 68.7% were men (55 individuals). Table 1 shows the characteristics of the group including age, height, weight, body mass index (BMI), number of days since stroke diagnosis, NIHSS score, sex, smoking, and history of chronic diseases.
OUTCOMES’ CHANGES AFTER REHABILITATION:
Figures 1–4 show a comparison of BI, mRS, vitamin D, and insulin-like growth factor 1 (IGF-1) results before and after rehabilitation. After therapy, there was a statistically significant increase in BI scores (median difference is 2.0 points; P<0.001) and IGF levels (median difference is 124.6 ng/ml; P<0.001). There was also a statistically significant reduction in mRS scores (median difference is 7.0 points; P<0.001). There were no significant differences in vitamin D levels (P=0.40).
VARIABLES AFFECTING OUTCOMES:
Table 2 shows the assessment of the impact of selected variables on activities of daily living (BI) and disability/dependence in the daily activities (mRS) of post-stroke patients (linear regression analysis in the univariate model). The effect of age (B=−0.01, P=0.04) and serum vitamin D levels (B=−0.02, P=0.01) on BI score was demonstrated. The effect of BMI results (B=−0.07, P=0.02) on mRS score was observed.
Firstly, a limitation of this study was that the trial was not randomized and it took place at a single center. In the future, it would be advisable to conduct a multicenter study for validation in other cohorts. We cannot directly relate our results to the above studies due to the fact that our study group included only patients after ischemic stroke but not treated with thrombolytic therapy. Future studies, which we plan to expand to a larger patient population, should take this aspect into account. Furthermore, there was no way to exclude errors related to variables that were not measurable in this observational study, such as sun exposure, diet, physical activity, or parathormone levels. No in-depth analysis of nutritional status was carried out and no information on nutritional conditions was collected. Future research should consider whether vitamin D supplementation in patients undergoing rehabilitation improves functional outcomes in ischemic stroke patients. The BI and mRS tools used are recommended for functional assessment and have high inter-rater reliability. Nevertheless, there is no mandatory definition of a good score, and it is the researcher who sets the standards that determine the score. Neither scale takes into account the patient’s mental state, and anxiety or post-stroke depression can affect a patient’s functioning and quality of life. Future studies should also use other objective measurement methods that take into account all aspects of life, both physical and mental.
In conclusion, lower serum vitamin D levels and older age may be associated with worse functional outcome in first-ever ischemic stroke patients. It is urgent to evaluate in a prospective, randomized trial with a placebo group whether vitamin D supplementation in appropriate doses can improve prognosis and functional outcomes in first-ever ischemic stroke patients undergoing rehabilitation treatment.
FiguresFigure 1. A comparison of BI results before and after rehabilitation. BI – Barthel Index. Figure 2. A comparison of mRS results before and after rehabilitation. mRS – modified Rankin scale. Figure 3. A comparison of IGF results before and after rehabilitation. IGF – insulin-growth factor. Figure 4. A comparison of vitamin D results before and after rehabilitation.
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