Sarcopenia and Vitamin D Deficiency in Patients with Crohn’s Disease: Pathological Conditions That Should Be Linked Together

Sarcopenia is a prevalent condition in patients with Crohn’s disease (CD), representing an independent predictor factor for the development of major postoperative complications. Thus, a proper assessment of the muscle strength, by using different validated tools, should be deemed an important step of the clinical management of these patients. Patients with CD are frequently malnourished, presenting a high prevalence of different macro- and micro-nutrient deficiencies, including that of vitamin D. The available published studies indicate that vitamin D is involved in the regulation of proliferation, differentiation, and regeneration of muscle cells. The relationship between vitamin D deficiency and sarcopenia has been extensively studied in other populations, with interesting evidence in regards to a potential role of vitamin D supplementation as a means to prevent and treat sarcopenia. The aim of this review was to find studies that linked together these pathological conditions.


Introduction
Crohn's disease (CD) is a chronic and progressive inflammatory bowel disease (IBD) that has a high impact on a patient's quality of life. It is well known that all segments of the gastrointestinal tract can be affected by CD, mainly the terminal ileum and colon. Inflammation is generally segmental, asymmetrical, and transmural [1]. Although progress has been made to achieve prolonged remission, almost half of the patients over time will develop complications (i.e., strictures, fistulas, and abscesses) that require surgical treatments [2]. The pathogenesis of CD is not yet fully understood, however, it clearly involves multiple factors, i.e., genetic susceptibility, environmental factors, and intestinal microflora, resulting in dysregulation of multiple and overlapping immune pathways [1].
In the last decades, the prevalence of CD has increased continuously worldwide, especially in the developed countries, primarily due to environmental factors, such as changes in dietary patterns and alterations in body composition [1]. Among these, the loss of muscle mass resulting in a decrease of muscle strength, a condition named sarcopenia, is an increasingly prevalent condition in patients with CD and is a strong independent predictor factor for the appearance of major postoperative complications [3].

The Assessment of Sarcopenia
Due to the well-established negative impact of sarcopenia on several pathological conditions, it should be mandatory for every clinician in charge of patients affected by chronic diseases, to evaluate and exclude the presence of sarcopenia, by means of validated case-finding tools.
Among these, the SARC-F questionnaire is the most used in daily clinical practice for patients aged ≥ 65 [12]. It is a self-reported questionnaire, with low sensitivity but high specificity, based on the patient's self-evaluation of five motor abilities, i.e., walking, rising from a chair, climbing stairs, carrying weights, and avoiding falls [12]. An alternative recommended case-finding tool is the Ishii screening test, which relates age, handgrip strength, and calf circumference [13]. Nevertheless, including a skeletal muscle strength evaluation could be considered a more reliable tool to detect sarcopenia.
Should the screening tests return positive, evidence of low muscle quantity or low muscle quality by the use of tools available to this purpose would confirm a formal diagnosis of sarcopenia. In clinical practice, tool selection may depend on several variables related to both the patient and healthcare setting [8]. A brief overview of these tools is presented in Table 1.

Sarcopenia in Patients with CD
In the last decade, sarcopenia has emerged as a primary factor in the nutritional assessment of patients affected by chronic inflammatory diseases, including IBD [14,15]. In fact, there is evidence indicating that this syndrome impacts the course of the disease, the responsiveness to specific therapies, and the outcomes of surgery [14].
Sarcopenia turns out to be a widespread condition in patients with IBD, in particular CD [16]. In a recent systematic review, it is reported that up to 60% of patients with IBD present a depletion of the muscle mass when compared with healthy subjects [16].
It is reported that patients with CD, affected by sarcopenia, result to be overweight or obese (a condition named "sarcopenic obesity"), rather than undernourished, at the nutritional assessment tests [17]. This extreme variability emphasizes the need for malnutrition and sarcopenia screening in all CD patients. In addition to malabsorption and gastrointestinal surgery, other factors may contribute to the development of sarcopenia in patients with CD, such as eventual glucocorticoid treatment and hypogonadism, and a reduced physical activity [18][19][20]. It should be noted that the activation of inflammatory cytokines may contribute significantly to converting the muscle protein metabolism from synthesis to degradation, as shown in Figure 1 [18,21].
To investigate the connection between sarcopenia and CD, we performed a systematic review, according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) checklist [22], based on the following Medical Subject Heading (MeSH) keywords: "Crohn's disease", "inflammatory bowel disease", "sarcopenia". The search, performed on 10 March 2021, was made on the following on-line databases: Scopus (www. scopus.com) (1969-2020), MEDLINE (www.nlm.nih.gov) (1969-2020), and the US National Library of Medicine (www.PubMed.gov). Two authors (F.P. and R.D.T.) performed the screening of titles and abstracts. Full-length versions of selected articles were then assessed for inclusion criteria: studies in patients aged >18 years; diagnosis of CD performed according to the current international guidelines; papers published in English; studies on IBD in which data on CD were clearly defined; studies in which the prevalence of sarcopenia was clearly quantified. The study selection process is presented in Figure 2. The following data were collected: number of patients; variable, test, and tool used to assess sarcopenia in patients with CD; percentage of patients with sarcopenia (Table 2). The combined effect of inflammatory cytokines (e.g., TNF-α, IL-6) in determining increased muscle protein degradation, through NF-KB and increased Myostatin activity, are here summarized. By reducing IGF-1 and its related intracellular signal pathway, along with the decreased amino acid absorption also due to therapeutic interventions, chronic inflammation determines a diminished protein synthesis. Abbreviations: TNF-α: tumor necrosis factor Alfa; IL-6: interleukin 6; PI3K: phosphatidylinositol-3-kinase; AKT: protein kinase B; mTORC1: mammalian target of rapamycin complex; IGF-1: Insulin-Like Growth Factor-1; NF-KB: Nuclear Factor Kappa B.
To investigate the connection between sarcopenia and CD, we performed a systematic review, according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) checklist [22], based on the following Medical Subject Heading (MeSH) keywords: "Crohn's disease", "inflammatory bowel disease", "sarcopenia". The search, performed on 10 March 2021, was made on the following on-line databases: Scopus (www.scopus.com) (1969-2020), MEDLINE (www.nlm.nih.gov) (1969-2020), and the US National Library of Medicine (www.PubMed.gov). Two authors (F.P. and R.D.T.) performed the screening of titles and abstracts. Full-length versions of selected articles were then assessed for inclusion criteria: studies in patients aged >18 years; diagnosis of CD performed according to the current international guidelines; papers published in English; studies on IBD in which data on CD were clearly defined; studies in which the prevalence of sarcopenia was clearly quantified. The study selection process is presented in Figure 2. The following data were collected: number of patients; variable, test, and tool used to assess sarcopenia in patients with CD; percentage of patients with sarcopenia (Table 2). The combined effect of inflammatory cytokines (e.g., TNF-α, IL-6) in determining increased muscle protein degradation, through NF-KB and increased Myostatin activity, are here summarized. By reducing IGF-1 and its related intracellular signal pathway, along with the decreased amino acid absorption also due to therapeutic interventions, chronic inflammation determines a diminished protein synthesis. Abbreviations: TNF-α: tumor necrosis factor Alfa; IL-6: interleukin 6; PI3K: phosphatidylinositol-3-kinase; AKT: protein kinase B; mTORC1: mammalian target of rapamycin complex; IGF-1: Insulin-Like Growth Factor-1; NF-KB: Nuclear Factor Kappa B.
Records identified from databases (n = 109) Duplicate records removed (n = 65) Records screened (n = 44) Records excluded (n = 9) Letters, reviews, meta---analysis Reports assessed for eligibility (n = 35) Reports excluded: • Studies in patients aged <18 years (n = 10) • Studies on IBD in which data on CD are not clearly defined (n = 7) • Studies in which the prevalence of sarcopenia is not clearly quantified (n = 5) Studies included in review (n = 13) Identification Screening Included Figure 2. Flow diagram of the study selection process (adapted from PRISMA) [22].  It has to be underlined that not all the above-cited studies were primarily focusing on the assessment of sarcopenia in patients with CD, which was instead part of a more extensive analysis. A common finding emerging from these studies, which were performed in different areas of the world, is the high prevalence of sarcopenia in patients with CD. The cohorts of these studies were heterogeneous and the percentage of patients with sarcopenia ranged from 19% to 61.4%, among different study populations. However, when considering only the data from the studies which used the gold standard tools (CT and MRI), the prevalence of sarcopenia increases significantly, ranging from 31% to 61.4%. Lastly, no significant difference in sample size, between studies using more expensive and time-consuming techniques (CT and MRI) and those that used more affordable tools (BIA and Dyn), is noteworthy.
Despite these described data, in daily clinical practice, the assessment of sarcopenia is still considered a marginal issue. Indeed, the most recent guidelines on CD do not mention the term "sarcopenia" [35].

Definition of Vitamin D Deficiency
Vitamin D is known to be produced in the skin from sunlight exposure or derived from foods that naturally contain vitamin D. According to the Endocrine Society guidelines, Vitamin D deficiency is defined as a serum 25-hydroxyvitamin D (25(OH)D) below 20 ng/mL (50 nmol/L), and vitamin D insufficiency as a serum 25(OH)D of 21-29 ng/mL (525-725 nmol/L) [36]. However, among scientific societies worldwide there is no general agreement on normal serum levels of 25(OH)D. Screening for vitamin D deficiency is indicated for all patients considered at risk, while population screening is not recommended [37]. In clinical practice, the most used way to evaluate vitamin D status is to determine serum concentrations of circulating 25(OH)D, measured with a reliable assay [36].
Vitamin D deficiency is acknowledged as a global health issue [38]. In addition to playing a crucial role in calcium and phosphorus homeostasis to preserve bone health, several studies have demonstrated a pleiotropic effect in different physiological processes. In particular, it has been recognized as a regulator of the innate immune system, of cardiovascular and renal functions, of cancer progression [37], and is also involved in different acute and chronic diseases [39].

Prevalence of Vitamin D Deficiency in Patients with CD
In the last decades, several studies have established the presence of vitamin D deficiency in patients with IBD, suggesting its potential role in the pathogenesis of these autoimmune diseases.
As previously done for sarcopenia, we performed a systematic review to highlight the evidence that linked vitamin D deficiency to CD. The research was carried out according to the PRISMA checklist [22], on the online databases mentioned above and based on the following MeSH keywords: "Crohn's disease", "inflammatory bowel disease", "vitamin D", "cholecalciferol", "25-hydroxyvitamin D", "vitamin D deficiency", "vitamin D status". The analysis of the studies was performed by R.D.T. and F.P. The inclusion criteria were: studies in patients aged >18 years; diagnosis of CD performed according to the current international guidelines; diagnosis of vitamin D insufficiency or deficiency established for serum levels lower than 30 ng/mL; studies on IBD in which data on CD were clearly defined; studies in which the prevalence of vitamin D was clearly quantified; papers published in English. The following data were collected: number of patients; cut-off of 25(OH)D expressed in ng/mL adopted by the authors to define vitamin D deficiency; percentage of patients with vitamin D deficiency ( Table 3). The study selection process is presented in Figure 3. Table 3. Prevalence of Vitamin D deficiency in Crohn's Disease (CD).

Authors
Year n Age (Years)

Vitamin D Deficiency (%)
Janssen et al. [  As shown in Table 3, all studies demonstrated vitamin D deficiency in patients with CD, ranging from 10.5% [49,59] to 100% [64], the majority showing high prevalence values.
It must be acknowledged that the majority of the studies in which vitamin D status was assessed were conducted on patients with IBD. To perform our review, we selected the studies in which the percentage of CD patients was clearly defined. 25(OH)D serum levels were expressed in ng/mL.
It should be specified that some authors provided different percentages based on the seasonal variability, with a lower prevalence of vitamin D deficiency occurring in summer than in winter [41,57,65,66], as expected.
Furthermore, recent data suggest that free 25(OH)D concentrations may be a better indicator than total 25(OH)D for the assessment of vitamin D status in patients with CD, due to the regulatory effects of glucocorticoid therapy and cytokines on vitamin D binding protein (VDBP) synthesis [70]. Reports excluded: • Studies in patients aged <18 years (n = 37) • In---vitro studies (n = 47) • Studies in pregnant woman (n = 1) • Studies on IBD in which data on CD are not clearly defined (n = 34) • Studies in which the prevalence of Vitamin D deficiency is not clearly quantified (n= 56) • Full---text not available (n=20) • Studies in languages other than English (n = 9) Studies included in review (n =30) Identification Screening Included Figure 3. Flow diagram of the study selection process (adapted from PRISMA) [22].

Effects of Vitamin D on Skeletal Muscle Function
Over recent years, the potential role of vitamin D on muscle function and strength has been widely debated [4].
At a cellular level, it is known that vitamin D acts through both genomic and nongenomic pathways, as summarized in Figure 4. At the nuclear level, vitamin D can regulate gene expression by interacting with Vitamin D Receptor (VDR), thus forming a heterodimeric complex of liganded VDR with Retinoid-X-receptor (RXR) and up-regulating or down-regulating target genes transcription. The non-genomic effects of Vitamin D are mediated by the activation of intracellular signal pathways through signal molecules, e.g., phospholipase C and phospholipase A2, and the production of second messengers, protein kinases, and the opening of Ca 2+ and Cl − channels as depicted in Figure 4. up-regulating or down-regulating target genes transcription. The non-genomic effects of Vitamin D are mediated by the activation of intracellular signal pathways through signal molecules, e.g., phospholipase C and phospholipase A2, and the production of second messengers, protein kinases, and the opening of Ca 2+ and Cl − channels as depicted in Figure 4. Focusing on the biological mechanisms that regulate differentiation, proliferation, and regeneration of muscle cells, it has been demonstrated that vitamin D regulates several myogenic transcription factors involved in muscle cells proliferation, e.g., insulin-like growth factor 2 and follistatin [71], and in muscle cells differentiation, e.g., fetal myosin, the neural cell adhesion molecule, insulin-like growth factor 1, fibroblast growth factor and myogenic differentiation protein 1 [72,73].
Regarding muscle regeneration, it has been demonstrated that vitamin D promotes the initial increase of the cross-sectional area of skeletal muscle fibers, by arresting the cell cycle, and suppresses the expression of myostatin, a key factor implicated in muscular degeneration [74].
According to the current knowledge, vitamin D seems to mainly affect type IIA muscle cells, i.e., the "fast twitch oxidative" cells [71]. Indeed, by using muscle biopsy, in previous works it has been shown that vitamin D deficiency is associated with type IIA muscle cells atrophy and fibrosis [75] and, by contrast, the supplementation of vitamin D has been shown to increase the number and the diameter of type IIA muscle cells, thus increasing muscle strength [76].
Furthermore, it should be mentioned that elevated PTH may contribute to the pathogenesis of sarcopenia, given its direct effect on skeletal muscle protein metabolism and the recent demonstration that elevated PTH levels are associated with vitamin D deficiency in sarcopenia [77]. Focusing on the biological mechanisms that regulate differentiation, proliferation, and regeneration of muscle cells, it has been demonstrated that vitamin D regulates several myogenic transcription factors involved in muscle cells proliferation, e.g., insulin-like growth factor 2 and follistatin [71], and in muscle cells differentiation, e.g., fetal myosin, the neural cell adhesion molecule, insulin-like growth factor 1, fibroblast growth factor and myogenic differentiation protein 1 [72,73].
Regarding muscle regeneration, it has been demonstrated that vitamin D promotes the initial increase of the cross-sectional area of skeletal muscle fibers, by arresting the cell cycle, and suppresses the expression of myostatin, a key factor implicated in muscular degeneration [74].
According to the current knowledge, vitamin D seems to mainly affect type IIA muscle cells, i.e., the "fast twitch oxidative" cells [71]. Indeed, by using muscle biopsy, in previous works it has been shown that vitamin D deficiency is associated with type IIA muscle cells atrophy and fibrosis [75] and, by contrast, the supplementation of vitamin D has been shown to increase the number and the diameter of type IIA muscle cells, thus increasing muscle strength [76].
Furthermore, it should be mentioned that elevated PTH may contribute to the pathogenesis of sarcopenia, given its direct effect on skeletal muscle protein metabolism and the recent demonstration that elevated PTH levels are associated with vitamin D deficiency in sarcopenia [77].

Vitamin D and Sarcopenia: Evidence from Other Patients
The relationship between vitamin D deficiency and sarcopenia has been extensively studied in other populations, and results from studies in these populations are interesting in regards to the potential role of vitamin D supplementation for the prevention and treatment of sarcopenia.
In particular, in the geriatric population, known to be at a high prevalence of vitamin D deficiency worldwide [78], a relationship between vitamin D and neuromuscular performance has been established [71,79].
Although the effects of the vitamin D on the intracellular nuclear and non-nuclear receptors to stimulate the growth and function of skeletal muscle cells has been well demonstrated in several studies, the clinical usefulness of oral vitamin D supplementation as a therapeutic mean to treat or prevent sarcopenia in older patients is still controversial [74].
This could interestingly be related to a decline in the VDR number with advancing age [80,81], thus making vitamin D supplementation probably more effective in younger patients.
It is worth noting that this specific issue has been the subject of a recent review within this journal and therefore will not be extensively discussed here [74].

The Missing Step: The Effect of Vitamin D Supplementation on Sarcopenia in Patients with CD
Our research aimed at finding relevant information in the scientific literature on the relationship between vitamin D supplementation and sarcopenia in patients with CD. However, in spite of the high prevalence of sarcopenia in patients with CD and the correlation between vitamin D metabolism and muscular performance, studies correlating these three clinical conditions are missing, although there is growing attention on this topic, especially among pediatric patients [82].
To the best of our knowledge, only Hradsky et al. performed a study on this issue, observing an improvement in muscle parameters after vitamin D supplementation in children with IBD, but without discrimination between CD and Ulcerative Colitis and using muscle strength as a variable to assess sarcopenia [83].
Given the role of vitamin D on muscle metabolism on a molecular basis, an improvement in sarcopenia in patients with CD could be expected with vitamin D supplementation. This open question requires further and appropriate studies.