ABSTRACT
Background and Aim
Zinc (Zn) deficiency occurs in 40% of patients with ulcerative colitis (UC). Although Zn‐dependent enzymes are involved in intestinal immune regulation, their relationships with Zn deficiency and mucosal inflammation remain unclear. This study aimed to investigate the associations among Zn‐dependent enzyme activities, serum Zn levels, and mucosal inflammatory cytokine mRNA expression in UC.
Methods
Patients with UC who underwent colonoscopies from September 2019 to October 2020 were registered in a single‐center prospective observational study. Serum Zn levels, serum and mucosal heat‐resistant alkaline phosphatase (hrALP) activity, serum and mucosal ecto‐5′‐nucleotidase (NT5E)/CD73 activity, and mucosal inflammatory cytokine mRNA expression were evaluated.
Results
Twenty‐nine patients with UC were enrolled. Serum Zn levels were lower in the nonclinical remission group and the non‐endoscopic remission group. Tissue hrALP activity was higher and tissue NT5E/CD73 activity tended to be higher in Zn‐deficient patients. Tissue hrALP was positively correlated with the Matts histological score, whereas tissue NT5E/CD73 activity showed a positive trend. Serum Zn levels were inversely correlated with tissue hrALP activity. Tissue hrALP and NT5E/CD73 activities were positively correlated with several mucosal inflammatory cytokines, whereas serum enzyme activities showed no significant correlations with inflammatory cytokine expression.
Conclusions
Increased mucosal Zn‐dependent enzyme activities were associated with histological inflammation, Zn deficiency, and mucosal inflammatory cytokine expression in UC. These findings suggest that mucosal Zn‐dependent enzyme activity may reflect local inflammatory status and altered Zn homeostasis in UC.
Keywords: ulcerative colitis, zinc deficiency, zinc‐dependent enzymes
1. Introduction
Ulcerative colitis (UC) is a major form of inflammatory bowel disease (IBD), and its prevalence has increased worldwide [1]. UC is characterized by chronic mucosal inflammation, starting in the rectum and extending throughout the colon. Mucosal inflammation induces mucosal erythema, erosions, and ulcers, thereby leading to abdominal pain, diarrhea, weight loss, and rectal bleeding [2, 3]. Malnutrition is observed in more than 60% of patients with UC [4]. Zinc (Zn) deficiency is one of the most common micronutrient deficiencies in patients with UC, affecting approximately 40% of patients [5]. Zn deficiency can cause diarrhea, impair immune function, and delay wound healing, potentially contributing to mucosal inflammation in UC [5]. Therefore, Zn supplementation has been considered a therapeutic strategy for Zn‐deficient patients with UC [6, 7].
Zn is an essential trace element and a cofactor for various enzymes involved in growth, immune function, and tissue repair [8, 9, 10]. Alkaline phosphatases (ALPs), including intestinal alkaline phosphatase (IAP) and ecto‐5′‐nucleotidase (NT5E)/CD73, are Zn‐dependent enzymes [11, 12, 13]. IAP maintains gut barrier function and modulates intestinal inflammation [11]. In UC, IAP mRNA expression is lower in the inflamed mucosa than in the non‐inflamed mucosa [14]. Reduced IAP expression may result in insufficient detoxification of lipopolysaccharide, thereby promoting mucosal inflammation [14]. Conversely, NT5E/CD73 plays a critical role in the production of adenosine, a significant anti‐inflammatory mediator [15, 16]. NT5E/CD73 expression is increased in UC compared with healthy controls [17]. Although Zn‐dependent enzyme activities may be influenced by serum Zn levels [12, 13, 16], their associations with disease severity and Zn deficiency in UC have not been fully elucidated. Furthermore, Zn and Zn‐dependent enzymes, including IAP and NT5E/CD73, may also modulate inflammatory cytokine responses, including interleukin (IL)‐1β, IL‐2, IL‐6, and tumor necrosis factor‐alpha (TNF‐α) [18, 19, 20]. In addition, Zn supplementation in Zn‐deficient patients with UC has been reported to reduce IL‐2 and IL‐6 levels [21]. However, the interplay between Zn deficiency, Zn‐dependent enzymes, and mucosal cytokine responses remains poorly understood in UC.
Therefore, we investigated the associations of heat‐resistant ALP (hrALP) activity, a surrogate measure enriched for IAP activity, and NT5E/CD73 activity with disease severity, serum Zn levels, and mucosal inflammatory cytokine expression in UC.
2. Materials and Methods
2.1. Study Population and Design
This prospective observational study was conducted at Asahikawa Medical University (AMU) Hospital. Patients with UC aged ≥ 12 years scheduled for colonoscopy (CS) from September 2019 to October 2020 were enrolled after providing written informed consent. Patients without available specimens or blood tests performed within 7 days before or after CS, as well as those with right‐sided UC, were excluded. This study was approved by the AMU Research Ethics Committee (No. 18245) and registered with UMIN (UMIN000036787). For the exploratory relapse analysis, patients in clinical remission were followed until March 2026.
2.2. UC Tissue Sample and Serum Collection
Rectal biopsy specimens (n = 30) were obtained during CS. Two specimens were placed in TRIzol (Invitrogen, Carlsbad, CA, USA) for RNA extraction, and the remaining two were snap‐frozen for enzyme assays. Serum samples were obtained from residual blood collected during routine clinical testing. Samples were stored at −80°C, and the snap‐frozen tissues and serum were shipped to Kyoto University for enzyme activity measurement. This protocol was approved by the Kyoto University Graduate School and Faculty of Medicine Ethics Committee (No. R2055).
2.3. Data Collection
Clinical data and blood test results (e.g., white blood cell [WBC], hemoglobin [Hb], platelet [Plt], albumin [Alb], C‐reactive protein [CRP], and Zn levels) were obtained from hospital records at the time of CS. A serum Zn level of ≥ 80 μg/dL was defined as sufficient, whereas < 80 μg/dL was deficient. Endoscopic and clinical activities were estimated using the Mayo endoscopic score (MES) and partial Mayo score (pMayo), respectively [22]. Endoscopic remission was defined as an MES of 0 or 1, and clinical remission as pMayo score ≤ 2 with a stool score of 0 or 1 and a rectal bleeding score of 0. Histological activity was assessed by the Matts classification [23]. Relapse was defined as a pMayo score > 2, treatment intensification, or loss of endoscopic remission.
2.4. Preparation of Lysates From the Excised Specimens
Biopsy tissues were homogenized in a lysis buffer (10‐mM Tris–HCl; pH 7.5; 0.1% Nonidet P‐40) and centrifuged at 2300 × g for 5 min at 4°C. The supernatant was used for enzyme activity assays.
2.5. Measurement of ALP Activity
Tissue lysates (5 μg) or serum (6 μL) were preincubated for 10 min at room temperature. A 100‐μL substrate solution (2 mg/mL disodium p‐nitrophenylphosphate hexahydrate [Wako Pure Chemicals Industries Ltd., Osaka, Japan] in 1‐M diethanolamine buffer, pH 9.8, containing 0.5 mM MgCl2) was added and incubated for 60 min at room temperature. The released p‐nitrophenol was quantified by absorbance at 405 nm using a Synergy H1 microplate reader (BioTek, Winooski, VT, USA). For measurement of hrALP, samples were incubated at 50°C for 30 min. hrALP activity was used as a surrogate measure enriched for IAP activity, although it is not specific for IAP alone.
2.6. Measurement of NT5E/CD73 Activity
NT5E/CD73 activity was measured using a 5′‐nucleotidase assay kit (Diazyme Laboratories, Poway, CA, USA) with 5 μg of rectal tissue lysates or 6 μL of serum, according to the manufacturer's protocol. Absorbance was measured at 550 nm using a Synergy H1 microplate reader (BioTek).
2.7. Measurement of Cytokine mRNA Expressions
Total RNA was extracted from rectal biopsy specimens using TRIzol (Invitrogen) and purified with an RNeasy mini kit (Qiagen, Hilden, Germany), according to the manufacturers' instructions. Complementary DNA (cDNA) was synthesized using a High‐capacity cDNA RT kit (Thermo Fisher Scientific, Waltham, MA, USA). Quantitative real‐time PCR was performed using an Applied Biosystems 7300 Real‐Time PCR system with TaqMan gene expression assays (Thermo Fisher Scientific) in duplicate (IL‐1β; Hs01555410, IL‐6; Hs00174131, IL‐8; Hs00174103, IL‐10; Hs00961622, IL‐12B; Hs01011518, IL‐17F; Hs01028648, IL‐23A; Hs00372324, TNF‐α; Hs00174128, IFN‐γ; Hs00989291).
2.8. Statistical Analysis
Statistical analyses were performed using GraphPad Prism 10.2.3 (GraphPad Software Inc., La Jolla, CA, USA). Categorical variables were compared using the chi‐squared test or Fisher's exact test, as appropriate. Continuous variables were compared using the Mann–Whitney U test, Wilcoxon matched‐pairs signed‐rank test, or unpaired t‐test. Univariate logistic regression analysis was performed to explore factors associated with Zn deficiency. Variables with p < 0.05 in univariate analysis were included in the multivariate logistic regression model. Correlations were calculated using Spearman's correlation coefficient. For exploratory relapse‐free survival analysis, patients in clinical remission were stratified by serum Zn level (< 80 vs. ≥ 80 μg/dL) or median tissue hrALP and NT5E/CD73 activities. Relapse‐free survival was estimated using the Kaplan–Meier method and log‐rank test. All reported p‐values of < 0.05 were considered statistically significant.
3. Results
3.1. Clinical Characteristics of the Patients With UC
The characteristics of the patients with UC are summarized in Table 1. Twenty‐nine patients with UC were included after exclusion of one patient with infectious colitis. The median age was 42 (interquartile range [IQR], 32–56.5) years. The median disease duration was 84 (IQR, 15–169.5) months. Disease extent was rectal in 6 (20.7%), left‐sided in 4 (13.8%), and extensive in 19 patients (65.5%). The median serum Zn was 82 (IQR, 68–93) μg/dL. Treatment included 5‐aminosalicylic acid in 28 patients (96.6%), corticosteroids in 4 (13.8%), immunomodulators (azathioprine or 6‐mercaptopurine) in 7 (24.1%), and biologics in 4 (13.8%). None received Zn supplementation.
TABLE 1.
Clinical characteristics of patients with ulcerative colitis.
| n = 29 | ||
|---|---|---|
| Age (year) | Median (IQR) | 41 (32–56.5) |
| Sex | ||
| Male | N (%) | 15 (51.7) |
| Female | N (%) | 14 (48.3) |
| Disease duration (month) | Median (IQR) | 84 (15–169.5) |
| Type of disease (disease extent) | ||
| E1 | N (%) | 6 (20.7) |
| E2 | N (%) | 4 (13.8) |
| E3 | N (%) | 19 (65.5) |
| pMAyo score | Median (IQR) | 1 (0.5–3.5) |
| MES | Median (IQR) | 1 (0–2) |
| Matts histological score | Median (IQR) | 2 (2–3) |
| WBC count | Median (IQR) | 5720 (4850–6990) |
| Hb (g/dL) | Median (IQR) | 14.1 (11.15–15.5) |
| Plt count | Median (IQR) | 26.8 (20–33.3) |
| CRP (mg/dL) | Median (IQR) | 0 (0–0.345) |
| Alb (g/dL) | Median (IQR) | 4.4 (4–4.65) |
| Zn (μg/dL) | Median (IQR) | 82 (68–93) |
| Concomitant medication | ||
| 5‐ASA | N (%) | 28 (96.6) |
| Corticosteroid | N (%) | 4 (13.8) |
| Azathioprine/6‐mercaptopurine | N (%) | 7 (24.1) |
| Calcineurin inhibitor | N (%) | 0 (0.0) |
| Biologics | N (%) | 4 (13.8) |
3.2. Comparison Between Zn‐Sufficient and Zn‐Deficient Patients With UC
The comparison between the Zn‐sufficient (Zn‐Suf, ≥ 80 μg/dL; n = 17) and Zn‐deficient (Zn‐Def, < 80 μg/dL; n = 12) groups is presented in Table 2. Zn deficiency was more prevalent in females (p = 0.025). The Zn‐Suf group had longer disease duration (126.0 vs. 22.5 months, p = 0.024). The Zn‐Def group showed higher disease activity with elevated pMayo, MES, and histological score (3.5 vs. 1, p < 0.001; 2 vs. 0, p = 0.003; and 3 vs. 2, p < 0.001, respectively). Blood parameters also differed significantly: WBC (6715 vs. 5104/mm3, p = 0.024), Hb (11.8 vs. 15 g/dL, p < 0.001), CRP (0.35 vs. 0 mg/dL, p = 0.021), and Alb (4.05 vs. 4.6 g/dL, p < 0.001). The median serum Zn levels were 89 (IQR, 83–98) and 62.5 (IQR, 53.5–71) μg/dL in the Zn‐Suf and Zn‐Def groups, respectively. Corticosteroid use was significantly higher in the Zn‐Def group than in the Zn‐Suf group (4 [33.3%] vs. 0 [0%], p = 0.0208). Exploratory univariate logistic regression analysis identified female sex, shorter disease duration, higher Matts histological score, lower albumin level, and higher tissue hrALP activity as factors associated with Zn deficiency. However, none of these variables remained statistically significant in multivariate analysis (Table S1).
TABLE 2.
Comparison between the zinc (Zn)‐sufficient and Zn‐deficient groups.
| Zn‐Suf group (n = 17) | Zn‐Def group (n = 12) | p | ||
|---|---|---|---|---|
| Age (year) | Median (IQR) | 48 (38–56.5) | 34 (24.75–60.25) | 0.1246 |
| Sex | ||||
| Male | N (%) | 12 (70.6) | 3 (25.0) | 0.0253 |
| Female | N (%) | 5 (29.4) | 9 (75.0) | |
| Disease duration (month) | Median (IQR) | 126 (36.5–200.0) | 22.5 (4.5–93) | 0.0243 |
| Type of disease (disease extent) | ||||
| E1 | N (%) | 4 (23.5) | 2 (16.7) | 0.6359 |
| E2 | N (%) | 3 (17.6) | 1 (8.3) | |
| E3 | N (%) | 10 (58.9) | 9 (75.0) | |
| pMAyo score | Median (IQR) | 1 (0–2) | 3.5 (1.25–6.75) | 0.0008 |
| MES | Median (IQR) | 0 (0–1) | 2 (1.25–2.75) | 0.0032 |
| Matts histological score | Median (IQR) | 2 (1–2) | 3 (2.25–4.75) | 0.0004 |
| WBC count | Median (IQR) | 5104 (4340–6630) | 6715 (5358–7493) | 0.0243 |
| Hb (g/dL) | Median (IQR) | 15 (14.1–15.95) | 11.8 (10.85–12.68) | 0.0005 |
| Plt count | Median (IQR) | 26.5 (20.15–30.35) | 33.3 (18.88–44.25) | 0.1985 |
| CRP (mg/dL) | Median (IQR) | 0 (0–0.1050) | 0.3450 (0–3.043) | 0.0211 |
| Alb (g/dL) | Median (IQR) | 4.6 (4.35–4.7) | 4.05 (2.8–4.2) | 0.0009 |
| Zn (μg/dL) | Median (IQR) | 89 (83–98) | 62.5 (53.5–71) | < 0.0001 |
| Concomitant medication | 0.302 | |||
| 5‐ASA | N (%) | 17 (100.0) | 11 (91.7) | 1 |
| Corticosteroid | N (%) | 0 (0.0) | 4 (33.3) | 0.0602 |
| Azathioprine/6‐mercaptopurine | N (%) | 4 (23.5) | 3 (25.0) | 1 |
| Calcineurin inhibitor | N (%) | 0 (0.0) | 0 (0.0) | 1 |
| Biologics | N (%) | 2 (11.8) | 2 (16.7) | 1 |
3.3. Association Between Zn Deficiency and Remission Rates in UC
Serum Zn levels were associated with clinical and endoscopic remission (Figure 1). The Zn‐Def group showed markedly lower clinical and endoscopic remission rates than the Zn‐Suf group (27.3% vs. 77.8% and 17.6% vs. 82.4%, respectively) (Figure 1A,B). Serum Zn levels were significantly lower in the nonclinical remission and non‐endoscopic remission groups (70.0 vs. 83.5 μg/dL, p = 0.010; 62.5 vs. 85.0 μg/dL, p = 0.001, respectively) (Figure 1C,D).
FIGURE 1.
Low clinical and endoscopic remission rates and high disease severity in zinc (Zn)‐deficient patients with ulcerative colitis (UC). Comparison of (A) clinical remission rates and (B) endoscopic remission rates between Zn‐sufficient (Zn‐Suf) and Zn‐deficient (Zn‐Def) patients with UC. Serum Zn levels in the (C) clinical remission and nonclinical remission groups and in the (D) endoscopic remission and non‐endoscopic remission groups. In subparts (C) and (D), *p < 0.05; **p < 0.01. Bars represent the median values and 25%–75% quartiles.
3.4. Inflammatory Cytokine Profile Between Zn‐Sufficient and Zn‐Deficient Patients With UC
The differences in cytokine mRNA expressions between the Zn‐Suf and Zn‐Def groups are shown in Figure 2. Most inflammatory cytokines were significantly higher in the Zn‐Def group, whereas IL‐12 was higher (p = 0.053) (Figure 2). Cytokine mRNA expression was also compared according to clinical and endoscopic remission status. IL‐1β, IL‐6, IL‐8, IL‐10, and IFN‐γ mRNA expressions were significantly higher, whereas IL‐17 was higher in the nonclinical remission group (Figure S1). As with the Zn‐Def group, in the non‐endoscopic remission group, the mRNA expressions of IL‐1β, IL‐6, IL‐8, IL‐10, IL‐17, IL‐23, TNF‐α, and IFN‐γ were significantly higher, whereas that of IL‐12 was relatively high (p = 0.1068) (Figure S2).
FIGURE 2.
Comparison of cytokine mRNA expressions (interleukin [IL]‐1β, IL‐6, IL‐8, IL‐10, IL‐17, IL‐23, interferon‐gamma [IFN‐γ], IL‐12, and tumor necrosis factor‐alpha [TNF‐α]) between Zn‐sufficient (Zn‐Suf) and Zn‐deficient (Zn‐Def) patients with UC. ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001. Bars represent the median values and 25%–75% quartiles.
3.5. High Tissue hrALP and NT5E/CD73 Activities in Nonremission and Zn‐Deficient Patients With UC
The activities of Zn‐dependent enzymes, including total ALP (tALP), hrALP, and NT5E/CD73, in serum and rectal tissues, are shown in Figures 3 and 4. Tissue hrALP activity was significantly higher, and tissue NT5/CD73 activity was relatively high in the nonclinical (7.10 vs. 32.11 mU/μg, p = 0.005; 9.45 vs. 12.7 mU/μg, p = 0.098, respectively) and non‐endoscopic remission groups (6.85 vs. 39.99 mU/μg, p < 0.001; 9.7 vs. 13.45 mU/μg, p = 0.054, respectively). Serum enzyme activities showed no significant difference between the groups (Figure 3A–D). Tissue hrALP activity was positively correlated with the Matts histological score (r = 0.6845, p < 0.0001), whereas tissue NT5/CD73 activity showed a nonsignificant trend toward a positive correlation (r = 0.3276, p = 0.0827) (Figure 3E,F). Tissue hrALP activity was significantly higher, and tissue NT5E/CD73 activity was higher in the Zn‐Def group (7.36 vs. 35.12 mU/μg, p = 0.006; 9.7 vs. 12.7 mU/μg, p = 0.07, respectively). Serum enzyme activities showed no significant difference between the groups (Figure 4A,B). Consistent with these findings, serum Zn levels were inversely correlated with tissue hrALP activity (r = −0.4667, p = 0.0107) (Figure 4C). Tissue NT5E/CD73 activity also tended to be inversely correlated with serum Zn levels (r = −0.3664, p = 0.0506) (Figure 4D).
FIGURE 3.
Zn‐dependent enzyme activities in patients with UC who are on remission and non‐remission. The activities of serum total ALP (tALP) and serum heat‐resistant ALP (hrALP) in the (A) clinical remission and nonclinical remission groups and in the (B) endoscopic remission and non‐endoscopic remission groups. Serum and tissue NT5E/CD73 activities in the (C) clinical remission and nonclinical remission groups and in the (D) endoscopic remission and non‐endoscopic remission groups. Correlation between (E) tissue hrALP activity and the Matts histological score. Correlation between (F) tissue NT5E/CD73 activity and the Matts histological score. ns, not significant; **p < 0.01; ****p < 0.0001. Bars represent the median values and 25%–75% quartiles.
FIGURE 4.
Zn‐dependent enzyme activities in Zn‐Suf and Zn‐Def patients with UC. (A) The activities of serum tALP, serum hrALP, and tissue hrALP in the Zn‐Suf and Zn‐Def groups. (B) Serum and tissue NT5E/CD73 activities in the Zn‐Suf and Zn‐Def groups. (C) Correlation between serum Zn levels and tissue hrALP activity. (D) Correlation between serum Zn levels and tissue NT5E/CD73 activity. ns, not significant; **p < 0.01. Bars represent the median values and 25%–75% quartiles.
3.6. Correlations Between Zn‐Dependent Enzymes, hrALP and NT5E/CD73, and Inflammatory Cytokines in Patients With UC
The correlations between inflammatory cytokines and Zn‐dependent enzymes are depicted in Figures 5 and S3–S5. Tissue hrALP activity was significantly positively correlated with IL‐1β, IL‐8, IL‐10, IL‐17, IL‐23, TNF‐α, and IFN‐γ, relatively positively correlated with IL‐6, and not significantly correlated with IL‐12 (Figure 5A). Furthermore, tissue NT5E/CD73 activity was significantly positively correlated with IL‐1β, IL‐10, IL‐12, IL‐17, IL‐23, TNF‐α, and IFN‐γ, relatively positively correlated with IL‐8, and not significantly correlated with IL‐6 (Figure 5B). In contrast, serum enzyme activities showed no significant correlations with any cytokines (Figures S3–S5).
FIGURE 5.
(A) Correlations between inflammatory cytokine mRNA expressions and tissue hrALP activities. (B) Correlations between inflammatory cytokine mRNA expressions and tissue NT5E/CD73 activities.
3.7. Exploratory Analysis of Relapse‐Free Survival in UC Patients With Clinical Remission
Among 18 patients in clinical remission at sample collection, six (33.3%) experienced relapse during a median follow‐up of 1738 days (IQR, 849–2212 days). Kaplan–Meier analysis demonstrated significantly lower relapse‐free survival in patients with serum Zn levels < 80 μg/dL than in those with serum Zn levels ≥ 80 μg/dL (p = 0.001). In contrast, relapse‐free survival did not differ significantly according to tissue hrALP activity (p = 0.458) or tissue NT5E/CD73 activity (p = 0.325) (Figure S6).
4. Discussion
To our knowledge, this is the first prospective study to evaluate the relationships among serum Zn deficiency, mucosal Zn‐dependent enzyme activities, and inflammatory cytokine expression in UC.
Zn deficiency in UC has been associated with mucosal inflammation, colitis exacerbation, and increased inflammatory cytokine production [24]. In this cohort, 12 of 29 patients with UC (41.3%) had Zn deficiency (Table 2), consistent with a previous study [5]. Zn deficiency has been reported to impair the activity of circulating Zn‐dependent enzymes, including ALP and NT5E/CD73 [16]. In contrast, previous studies in inflamed UC mucosa have shown reduced epithelial IAP expression but increased NT5E/CD73 expression [14, 17]. In our study, mucosal hrALP and NT5E/CD73 activities were increased in active UC, especially Zn‐deficient patients, whereas serum enzyme activities did not differ significantly (Figure 4). In addition, serum Zn levels were inversely correlated with tissue hrALP activity, whereas tissue NT5E/CD73 activity showed a trend toward an inverse correlation. These findings suggest that regulation of Zn‐dependent enzymes may differ between systemic circulation and the local intestinal mucosa and raise the possibility that alterations in mucosal Zn‐dependent enzyme activity may be associated with Zn homeostasis and may not be merely secondary markers of inflammation.
The apparent discrepancy between reduced epithelial IAP expression in inflamed UC mucosa and increased mucosal hrALP activity observed in our study requires careful interpretation [14]. Our study measured mucosal hrALP activity, which was used as a surrogate measure enriched for IAP activity but is not specific for IAP alone, rather than epithelial IAP expression itself. Therefore, reduced epithelial IAP expression and increased mucosal hrALP activity are not necessarily contradictory. In inflamed mucosa, hrALP activity may be influenced by changes in cellular composition, inflammatory cell infiltration, post‐translational regulation, substrate availability, and other local inflammatory conditions. In addition, ALP has been proposed to exert protective effects in intestinal inflammation through detoxification of luminal bacterial lipopolysaccharide and modulation of mucosal immune responses [14]. From this perspective, the increased mucosal hrALP activity observed in our study may reflect, at least in part, a compensatory or protective functional response to ongoing mucosal inflammation. Importantly, the cross‐sectional nature of this study limits our ability to determine whether enhanced mucosal enzyme activity contributes to Zn deficiency or instead represents a consequence or compensatory response to ongoing inflammation. Larger longitudinal studies, including assessment before and after Zn supplementation, are needed to clarify these mechanisms.
Zn deficiency has also been reported in patients with UC in remission and has been associated with poor outcomes, including relapse rates and hospitalization [5, 25]. In our study, 27.3% of Zn‐deficient patients were in clinical remission (Figure 1A). Although poor intake and malabsorption may contribute to Zn deficiency in active UC [26], these mechanisms alone may not explain Zn deficiency in remission. Zn deficiency develops in patients with chronic inflammatory diseases, including rheumatoid arthritis, even without intestinal inflammation [8]. Chronic inflammation and persistent immune system activation elevate Zn requirements, which are modulated by Zn transporters expressed on inflammatory cells [8, 27]. Furthermore, inflammatory cytokines such as TNF‐α and IL‐6 may regulate serum Zn levels as part of the inflammatory response [28]. In our data, multiple mucosal cytokines were elevated in the non‐endoscopic remission group, whereas several cytokines, including IL‐12, IL‐23, and TNF‐α, did not differ between the clinical remission and nonclinical remission groups (Figures S1 and S2). This suggests that mucosal inflammation may persist even in some patients with clinical remission. Consistent with this concept, tissue hrALP activity was positively correlated with the Matts histological score, whereas tissue NT5E/CD73 activity showed a trend toward a positive correlation. These findings suggest that local Zn‐dependent enzyme activities may reflect histological inflammatory status in the intestinal mucosa. Although we assessed only two of the numerous Zn‐dependent enzymes, these findings raise the possibility that Zn deficiency may reflect residual inflammation in patients with clinical remission. Further studies of mucosal Zn transporters are needed. Furthermore, exploratory analysis suggested that serum Zn level may have prognostic value for relapse, whereas tissue hrALP and NT5E/CD73 activities were not significantly associated with relapse‐free survival. However, these findings require validation in larger prospective studies.
IAP and NT5E/CD73 may also be relevant biomarkers and therapeutic targets in IBD [14, 17, 29, 30]. In active IBD, CD73 is associated with a pro‐inflammatory Th17‐cell phenotype and may serve as a marker of disease activity [29]. Conversely, IAP administration has been shown to improve the inflammation in experimental colitis models [14]. Therefore, these findings support a role for these enzymes in intestinal inflammation. Because Zn is required for Zn‐dependent enzyme activities [11, 12, 13, 16], Zn supplementation has recently emerged as a pivotal therapeutic approach for IBD [6, 7, 21]. Zn supplementation may influence mucosal immune responses and barrier functions, including pathways involving these enzymes.
This study has several limitations. Medication exposure was highly unbalanced, limiting stratified analyses and reliable assessment of treatment‐specific effects. Exploratory multivariate analysis did not identify independent factors associated with Zn deficiency, likely because of the small sample size and the interrelated clinical variables; therefore, residual confounding by disease severity, nutritional status, medication exposure, sex, and disease duration cannot be excluded. In addition, the Zn‐deficient group included a higher proportion of female patients and had a shorter disease duration, which may have acted as confounding factors influencing disease activity and enzyme expression. Furthermore, the hrALP assay was not specific for IAP, and the findings should therefore be interpreted as reflecting hrALP activity rather than direct IAP‐specific activity. Larger longitudinal studies are needed to clarify the clinical relevance of these findings.
In conclusion, Zn deficiency was associated with increased mucosal hrALP and NT5E/CD73 activities and elevated mucosal inflammatory cytokine expression in UC. These findings suggest that mucosal Zn‐dependent enzyme activity may reflect local mucosal inflammation and altered Zn homeostasis. Zn deficiency may reflect residual mucosal inflammation even in clinical remission, although this interpretation remains hypothesis‐generating. Larger longitudinal studies, including evaluation before and after Zn supplementation, are needed to clarify causality and clinical relevance.
Funding
This work was supported by the Nobelpharma Co. Ltd. for the purchase of experimental agents and the delivery of samples to the collaborator.
Conflicts of Interest
M.F. has received honoraria and had expenses paid to attend or give a presentation or advice at a meeting for EA Pharma Co. Ltd., AYUMI Pharmaceutical Corporation, AbbVie GK, Otsuka Pharmaceutical Factory Inc., Zeria Pharmaceutical Co. Ltd., JIMRO Co. Ltd., Nippon Kayaku Co. Ltd., elpharma Co. Ltd., Pfizer Japan Inc., Janssen Pharmaceutical K.K., Kyorin Pharmaceutical Co. Ltd., Mochida Pharmaceutical Co. Ltd., Daiichi Sankyo Company, Limited, Mitsubishi Tanabe Pharma Corporation, Takeda Pharmaceutical Co. Ltd., Yakult Honsha Co. Ltd., Olympus Corporation, Celltrionhealthcare.jp, Alfresa Pharma Corporation, Mylan Inc., Boston Scientific Corporation, Covidien Japan Inc., FUJIFILM Corporation, Fuji Chemical Industries Co. Ltd., and JIMRO Co. Ltd. and received research grants from EA Pharma Co. Ltd., AYUMI Pharmaceutical Corporation, AbbVie GK, Otsuka Pharmaceutical Factory Inc., Zeria Pharmaceutical Co. Ltd., Nippon Kayaku Co. Ltd., Nobelpharma Co. Ltd., Pfizer Inc., Janssen Pharmaceutical K.K., Kyorin Pharmaceutical Co. Ltd., Mochida Pharmaceutical Co. Ltd., Daiichi Sankyo Company, Limited, Mitsubishi Tanabe Pharma Corporation, Takeda Pharmaceutical Co. Ltd., Yakult Honsha Co. Ltd., JIMRO Co. Ltd., and Kamui Pharma Inc.
Y.M., T.K., Y.K., A.S., K.A., N.U., and K.M. declare no conflicts of interest.
Supporting information
Table S1: Univariate and multivariate logistic regression analyses of factors associated with Zn deficiency in patients with UC.
Figure S1: Comparison of cytokine mRNA expressions (IL‐1β, IL‐6, IL‐8, IL‐10, IL‐17, IL‐23, IFN‐γ, IL‐12, and TNF‐α) between the clinical remission and nonclinical remission groups. *p < 0.05. Bars represent the median values and 25%–75% quartiles.
Figure S2: Comparison of cytokine mRNA expressions (IL‐1β, IL‐6, IL‐8, IL‐10, IL‐17, IL‐23, IFN‐γ, IL‐12, and TNF‐α) between the endoscopic remission and non‐endoscopic remission groups. ns, not significant; ***p < 0.001; ****p < 0.0001. Bars represent the median values and 25%–75% quartiles.
Figure S3: Correlations between inflammatory cytokine mRNA expressions and serum tALP activities.
Figure S4: Correlations between inflammatory cytokine mRNA expressions and serum hrALP activities.
Figure S5: Correlations between inflammatory cytokine mRNA expressions and serum NT5E/CD73 activities.
Figure S6: Kaplan–Meier curves of relapse‐free survival stratified by serum Zn level, tissue hrALP activity, and tissue NT5E/CD73 activity in patients with UC in clinical remission.
Data Availability Statement
The data underlying this article will be shared on reasonable request to the corresponding author.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Table S1: Univariate and multivariate logistic regression analyses of factors associated with Zn deficiency in patients with UC.
Figure S1: Comparison of cytokine mRNA expressions (IL‐1β, IL‐6, IL‐8, IL‐10, IL‐17, IL‐23, IFN‐γ, IL‐12, and TNF‐α) between the clinical remission and nonclinical remission groups. *p < 0.05. Bars represent the median values and 25%–75% quartiles.
Figure S2: Comparison of cytokine mRNA expressions (IL‐1β, IL‐6, IL‐8, IL‐10, IL‐17, IL‐23, IFN‐γ, IL‐12, and TNF‐α) between the endoscopic remission and non‐endoscopic remission groups. ns, not significant; ***p < 0.001; ****p < 0.0001. Bars represent the median values and 25%–75% quartiles.
Figure S3: Correlations between inflammatory cytokine mRNA expressions and serum tALP activities.
Figure S4: Correlations between inflammatory cytokine mRNA expressions and serum hrALP activities.
Figure S5: Correlations between inflammatory cytokine mRNA expressions and serum NT5E/CD73 activities.
Figure S6: Kaplan–Meier curves of relapse‐free survival stratified by serum Zn level, tissue hrALP activity, and tissue NT5E/CD73 activity in patients with UC in clinical remission.
Data Availability Statement
The data underlying this article will be shared on reasonable request to the corresponding author.