*2.2. Data Analysis*

Two investigators (RZ, AS) extracted the following information separately and in duplicate in piloted form: author, publication year, survey year, country, and design (longitudinal, cross-sectional). Researchers tabulated data by IBD type of disease (Crohn's disease, CD, and ulcerative colitis, UC) to retrieve information on (1) sample size (*n*), (2) age (expressed as mean ± standard deviation, SD, or interquartile range, IQR), (3) male and female representativeness (expressed as *n* and %), serum zinc levels (according to IBD type, where possible, and expressed as mean ± SD or median and IQR), (4) threshold value used to assess zinc deficiency, (5) prevalence of zinc deficiency by IBD, and (6) summary of study findings. All references selected for retrieval from the databases were managed with the MS Excel data collection software platform by an expert biostatistician (FC). Finally, the data extracted from the selected studies and stored in the database were structured as evidence tables.

The quality of the studies included in the meta-analysis was evaluated using a tool developed by Hoy and colleagues [13]. Each study was assigned a score of one (yes) or zero (no) for each of the ten criteria. Based on the total score, studies were categorized as having a low (>8), moderate (6–8), or high (≤5) risk of bias. Disagreements between the two investigators on the methodological quality of the included studies were addressed by discussion, involving a third investigator in the final agreement (RS). All data analyses were performed using R, version 2021.09.1; our biostatistician (FC) used the meta-package to conduct meta-analyses of the zinc deficiency prevalence (%), subdivided according to IBD illness type (CD, UC). A common-effects model was used to calculate the prevalence and 95% confidence intervals (CI) (Figures 1 and 2). The Higgins and colleagues [14] *I* <sup>2</sup> test was used to estimate percentage heterogeneity between studies that cannot be explained by chance. The closer this value is to zero, the less the variability between studies. Negative values are comparable to zero and indicate that there is no heterogeneity. Values below 25% suggest a low, between 25% and 50% moderate, and above 50% high heterogeneity among studies. The funnel plot shown in Figure 3 was used as a visual tool when investigating

publication bias. The horizontal axis shows the scatter of treatment effects estimated from individual studies, while the vertical axis shows study size.

**Figure 1.** Flow diagram of literature screening process.


**Figure 2.** Pooled and grouped prevalence of zinc deficiency in IBD [15–23].

**Figure 3.** Funnel plot for assessment of publication bias across selected studies. (*n* = 9).

#### **3. Results**

The first systematic search of the literature yielded 152 entries. After excluding duplicates, 75 were classified as potentially relevant and selected for the title and abstract analysis. Then, 37 were excluded for failure to meet the characteristics of the approach or the review goal. After reviewing the full text of the remaining 38 records, only 9 met the inclusion criteria and were included in the meta-analysis [15–23]. The Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) flow chart illustrating the number of studies at each stage of the review is shown in Figure 1. The final study base included nine articles reporting zinc deficiency prevalence by IBD condition (CD, UC).

Details of the design (cohort or cross-sectional), sample size (*n*) and gender ratio (%), survey year, study population, age range, serum zinc levels at recruitment, zinc deficiency cutoffs, country, and summary of findings are provided in Table 1. The crosssectional design (67%, *n* = 6) predominated over the longitudinal (33%, *n* = 3). Recruitment settings were all community-based, and the geographic distribution of studies favored Asia (67%, *n* = 6), followed by Europe (22%, *n* = 2) and America (11%, *n* = 1). Following the inclusion criteria, all subjects had IBD. Only one study investigated subjects with Crohn's disease alone, whereas the other eight analyzed both IBD conditions. In total, this meta-analysis analyzed 736 subjects suffering from UC and 1677 with CD, resulting in a total IBD population of 2413 subjects. About 46% of the CD population and ~47% of the UC population were female. The prevalence of zinc deficiency across the selected studies showed higher mean values in the CD than in the UC population. Clustered analyses by IBD subgroup (CD, UC) showed a total population of 1677 for CD, with an overall zinc deficiency mean prevalence of 54% (95%CI 0.51 to 0.56). Notably, within this subgroup, the largest reports by Ehrlich and colleagues [15] and Sakurai and colleagues [21] reported a higher prevalence of 88% (95%CI 0.83 to 0.92) and 86% (95%CI 0.82 to 0.90), respectively (Table 2). In contrast, the total UC population of 806 subjects showed an overall 41% prevalence (95%CI 0.38 to 0.45) of zinc deficiency. The prevalence was more evenly distributed within this subgroup, apart from in the MacMaster and colleagues study [17], which reported significantly higher numbers (77%, 95%CI 0.58 to 0.90). The meta-analysis produced an overall estimate of 50% (95%CI 0.48 to 0.52) zinc deficiency prevalence, with high heterogeneity *I* <sup>2</sup> = 96% (Table 2). The funnel plot analysis showed no evidence of publication bias (Figure 2). According to the 10-item quality assessment checklist for prevalence studies by Hoy and colleagues [13], we found a moderate (*n* = 5) [16–18,21,22] to low (*n* = 4) [15,19,20,23] risk of bias across selected studies (Table 1).




Abbreviations: CD (Crohn's Disease), UC (Ulcerative Colitis), CI (Confidence Interval).

#### **4. Discussion**

The present systematic review and meta-analysis aimed to provide a revised estimate of the prevalence of zinc deficiency in IBD populations, without restrictions as to the country, patients age, and study design. We clustered the 17 entries from the nine studies conducted in three different countries (Asia, Europe, and America) that had reported single prevalence data by type of IBD within the population examined. To the best of our knowledge, no meta-analytic report has yet been published in the literature on this topic. Given the growing concern about the management of individuals with chronic malabsorption diseases, zinc deficiency in CD and UC is among the major sensitive issues. As the main finding, this meta-analysis produced an overall zinc deficiency estimate of 50% (95%CI 0.48–0.52), with high heterogeneity *I* <sup>2</sup> = 96% and a moderate to low risk of bias across selected reports. Funnel plot analysis showed no evidence of publication bias. The clustered meta-analysis by the IBD group (CD and UC) showed a higher overall prevalence of zinc deficiency in the CD group than in the UC group (54%, 95%CI 0.51 to 0.56, versus 41%, 95%CI 0.38 to 0.45). In fact, while CD is known to affect any part of the gastrointestinal tract, including the mouth, esophagus, stomach, small and large intestines, rectum, and anus, UC compromises the colon and rectum. Moreover, the more malabsorptive CD affects all layers of the intestinal wall discontinuously, whereas in UC, the inflammation occurs in the innermost lining of the intestinal wall and is a continuous stretch within the colon.

Of the trace element deficiencies so far reported in the literature, zinc deficiency in IBD patients may result from poor oral intake and especially from the intrinsic malabsorptive nature of IBD. As proven by preclinical models and translational studies in humans, the relationship between trace zinc and chronic malabsorptive disease must be considered bidirectional since low serum zinc concentrations may also exacerbate inflammation through dysfunction and deficient epithelial barrier reconstruction, altered mucosal immunity, and increased pro-inflammatory cytokines [24–26]. In support of these mechanistic hypotheses, a recent report suggested that zinc supplementation might favor permeability modifications in CD patients in remission. Thus, an improved intestinal barrier function may help reduce the recurrence risk, especially in CD [9]. Indeed, the enhanced gastrointestinal epithelial barrier function driven by zinc may play an essential role In potential therapeutic actions, especially in CD, which is more malabsorptive than UC. This latter point is also corroborated by the known proximal intestinal sites of zinc absorption, i.e., the duodenum and jejunum [27].

Furthermore, previous research suggested that zinc may have some efficacy in modulating the immune system through an improved response to pathogens, reduced inflammatory response, and improved atopic/allergic reactions. Zinc is also involved in cell cycle regulation, particularly apoptosis, and hence has potential anticarcinogenic effects. All these effects have a "symbiotic" relationship with the gut microbiota [28]. From a prognostic perspective, zinc deficiency may predispose to growth retardation in young populations, and to loss of appetite, impaired immune function, and structural impairment of the intestinal endothelium. In severe cases, it may also drive hair loss, diarrhea, delayed sexual maturation, impotence, hypogonadism in males, and eye and skin lesions [29]. All these aspects highlight the importance of early nutritional preventive management in IBD settings, from better quality of life and healthcare burden perspectives. As to the loss of appetite reported in zinc deficiency [30], lower trace zinc values may also facilitate unintentional weight loss, malnutrition [31], sarcopenia, and cachectic states, mainly in restrictive dietary settings. With regard to the structural impairment of the intestinal endothelium, intervention studies on CD patients observed that zinc supplementation has some potential to reduce transmucosal leakage in these patients [9]. This latter finding is critical because intestinal epithelial barrier dysfunction may allow leukocytes to pass through, causing exposure to a "storm" of luminal antigens, a hallmark of IBD activity. Therefore, zinc supplementation may reduce the inflammatory response and maintain remission in CD. However, this empirical evidence is not yet supported by specific guidelines regulating supplementation [32].

In order to supplement the biological explanation of our prevalence data, we looked at information on inflammation in the selected reports, considering CRP values and disease activity scores in relation to zinc deficiency. On the one hand, we found considerable inconsistency in the data, as a minority of 33% indicated a significant correlation between zinc deficiency and elevated CRP [15,18,19,21], while a statistical relation or data collection was lacking for the remainder. On the other hand, a clearer trend was found for the activity score in relation to zinc deficiency, although the scores used were heterogeneous across reports. However, available data are still insufficient to comprehensively analyze the prevalence categorized by the activity score variable, although this covariate is certainly to be considered in the fluctuations of the prevalence of zinc deficiency in IBD.

The limited data and biographical heterogeneity of the study populations reduce the reliability of this meta-analysis in qualitative terms. In addition, designs differed among the selected studies: the cross-sectional was the most common. However, this is the first meta-analytic study to be conducted on the prevalence of zinc deficiency in IBD. Further investigation is needed to corroborate and better define these data and how they may fluctuate in relation to disease activity.

#### **5. Conclusions**

Prevention of micronutrient deficiencies has the potential to reduce the risk of diseaserelated disability. Still, more evidence is needed to corroborate these first prevalence metadata. The present research highlights the importance of considering zinc as a micronutrient to be monitored, because every second IBD patient shows a deficiency. According to our results, zinc deficiency is more prevalent in the CD population, probably due to the more severely malabsorptive nature of this condition and also in light of the proximal site of zinc absorption in the intestine. In light of this, the latest ESPEN micronutrient guidelines [33] point out that the dietary reference intakes (DRI) of zinc for adults should be 8–15 mg. However, in malabsorption conditions, such as short bariatric surgery, cystic fibrosis, chronic pancreatitis, and IBD, the need for higher amounts of zinc (30–40 mg daily) to maintain the zinc balance needs to be considered.

**Author Contributions:** R.Z., F.C., A.S. and R.S.: conceptualization, research, resource provision, data collection, writing original version, and visualization. R.S. and G.G.: review and correction. R.Z., S.D.N., G.D.P., M.M., M.C. and R.B.: research and data collection. R.S.: conceptualization, validation, review, and correction. R.S. and F.C.: conceptualization, validation, review and correction, and visualization. All authors have read and agreed to the published version of the manuscript.

**Funding:** The Italian Ministry of Health with "Ricerca Corrente 2020" funds.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding authors.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

