Long-Term Vitamin D Deficiency Results in the Inhibition of Cell Proliferation and Alteration of Multiple Gastric Epithelial Cell Lineages in Mice.

Over one billion people globally are vitamin D (VD) deficient. Studies on the biological roles of VD are numerous but very little on the stomach. This project aims to understand how gastric homeostasis is affected by VD deficiency caused by prolonged exposure to darkness alone or combined with VD deficient diet. Three groups of C57/BL6 mice were subjected to different light exposure conditions and diets for 12 months (n = 8−12/group): control­12 h/12 h light/dark SDL (Standard Diet/Light), 24 h dark SDD (Standard Diet/Dark), and 24 h dark VDD (VD deficient diet/Dark). Stomach samples were collected for different multi-label lectin-/immuno-histochemical and qRT-PCR analyses, and the serum for LC-MS-MS. We found that the membrane VD receptor is expressed widely in the stomach when compared to nuclear VD receptors. Compared to SDL, VDD mice developed mucous cell expansion with increased mucins-mRNA (3.27 ± 2.73 (p < 0.05)) increased apoptotic cells, 15 ± 7 (p ≤ 0.001)); decreased cell proliferation, 4 ± 4 (p < 0.05)) and decreased acid secretion 33 ± 2 µEq/kg (p ≤ 0.0001)). Interestingly, mice exposed to full darkness developed mild VD deficiency with higher VD epimer levels: 11.9 ± 2.08 ng/mL (p ≤ 0.0001)), expansion in zymogenic cell number (16 ± 3 (p ≤ 0.01)), and a reduction in acid secretion (18 ± 2 µEq/kg (p ≤ 0.0001)). In conclusion, changes in light exposure or VD levels have serious physiological effects on the gastric mucosa, which should be considered during the management of gastric disorders.

An LC-MS/MS Method for Analysis of Vitamin D Metabolites and C3 Epimers in Mice Serum: Oral Supplementation Compared to UV Irradiation.

INTRODUCTION: The most common forms of vitamin D in human and mouse serum are vitamin D3 and vitamin D2 and their metabolites. The aim of this study is to determine whether diet and sunlight directly affect the circulating concentrations of vitamin D metabolites in a mouse model. We investigated the serum concentrations of eight vitamin D metabolites-vitamin D (vitamin D3 + vitamin D2), 25OHD (25OHD3 + 25OHD2), 1α25(OH)2D (1α25(OH)2D2, and 1α25(OH)2D3)-including their epimer, 3-epi-25OHD (3-epi-25OHD3 and 3-epi-25OHD2), and a bile acid precursor 7alpha-hydroxy-4-cholesten-3-one (7αC4), which is known to cause interference in liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. METHOD: The LC-MS/MS method was validated according to FDA-US guidelines. The validated method was used for the analysis of mouse serum samples. Forty blood samples from mice were collected and divided into three groups. The first group, the DDD mice, were fed a vitamin D-deficient diet (25 IU VD3/kg of diet) and kept in the dark; the second group, the SDD mice, were maintained on a standard-vitamin D diet (1000 IU VD3) and kept in the dark; and the third group, SDL, were fed a standard-vitamin D diet (1000 IU VD3) but kept on a normal light/dark cycle. LC-MS/MS was used for the efficient separation and quantitation of all the analytes. RESULTS: The validated method showed good linearity and specificity. The intraday and interday precision were both <16%, and the accuracy across the assay range was within 100 ± 15%. The recoveries ranged between 75 and 95%. The stability results showed that vitamin D metabolites are not very stable when exposed to continuous freeze-thaw cycles; the variations in concentrations of vitamin D metabolites ranged between 15 and 60%. The overlapping peaks of vitamin D, its epimers, and its isobar (7αC4) were resolved using chromatographic separation. There were significant differences in the concentrations of all metabolites of vitamin D between the DDD and SDL mice. Between the groups SDD (control) and SDL, a significant difference in the concentrations of 3-epi-25OHD was noted, where C3 epimer was about 30% higher in SDL group while no significant differences were noted in the concentrations of vitamin D, 25OHD, 1α25(OH)2D, and 7αC4 between SDD and SDL group. CONCLUSIONS: A validated method, combined with a simple extraction technique, for the sensitive LC-MS/MS determination of vitamin D metabolites is described here. The method can eliminate the interferences in LC-MS/MS analysis caused by the overlapping epimer and isobar due to them having the same molecular weights as 25OHD. The validated method was applied to mouse serum samples. It was concluded that a standard-vitamin D diet causes an increase in the proportion of all the vitamin D metabolites and C3 epimers and isobar, while UV light has no pronounced effect on the concentrations of the majority of the vitamin D metabolites except 3-epi-25OHD. Further studies are required to confirm this observation in humans and to investigate the biochemical pathways related to vitamin D's metabolites and their epimers.

Epimers of Vitamin D: A Review.

In this review, we discuss the sources, formation, metabolism, function, biological activity, and potency of C3-epimers (epimers of vitamin D). We also determine the role of epimerase in vitamin D-binding protein (DBP) and vitamin D receptors (VDR) according to different subcellular localizations. The importance of C3 epimerization and the metabolic pathway of vitamin D at the hydroxyl group have recently been recognized. Here, the hydroxyl group at the C3 position is orientated differently from the alpha to beta orientation in space. However, the details of this epimerization pathway are not yet clearly understood. Even the gene encoding for the enzyme involved in epimerization has not yet been identified. Many published research articles have illustrated the biological activity of C3 epimeric metabolites using an in vitro model, but the studies on in vivo models are substantially inadequate. The metabolic stability of 3-epi-1α,25(OH)2D3 has been demonstrated to be higher than its primary metabolites. 3-epi-1 alpha, 25 dihydroxyvitamin D3 (3-epi-1α,25(OH)2D3) is thought to have fewer calcemic effects than non-epimeric forms of vitamin D. Some researchers have observed a larger proportion of total vitamin D as C3-epimers in infants than in adults. Insufficient levels of vitamin D were found in mothers and their newborns when the epimers were not included in the measurement of vitamin D. Oral supplementation of vitamin D has also been found to potentially cause increased production of epimers in mice but not humans. Moreover, routine vitamin D blood tests for healthy adults will not be significantly affected by epimeric interference using LC-MS/MS assays. Recent genetic models also show that the genetic determinants and the potential factors of C3-epimers differ from those of non-C3-epimers.Most commercial immunoassays techniques can lead to inaccurate vitamin D results due to epimeric interference, especially in infants and pregnant women. It is also known that the LC-MS/MS technique can chromatographically separate epimeric and isobaric interference and detect vitamin D metabolites sensitively and accurately. Unfortunately, many labs around the world do not take into account the interference caused by epimers. In this review, various methods and techniques for the analysis of C3-epimers are also discussed. The authors believe that C3-epimers may have an important role to play in clinical research, and further research is warranted.

A Narrative Role of Vitamin D and Its Receptor: With Current Evidence on the Gastric Tissues.

Vitamin D is a major steroid hormone that is gaining attention as a therapeutic molecule. Due to the general awareness of its importance for the overall well-being, vitamin D deficiency (VDD) is now recognized as a major health issue. The main reason for VDD is minimal exposure to sunlight. The vitamin D receptor (VDR) is a member of the steroid hormone receptors that induces a cascade of cell signaling to maintain healthy Ca2+ levels that serve to regulate several biological functions. However, the roles of vitamin D and its metabolism in maintaining gastric homeostasis have not yet been completely elucidated. Currently, there is a need to increase the vitamin D status in individuals worldwide as it has been shown to improve musculoskeletal health and reduce the risk of chronic illnesses, including some cancers, autoimmune and infectious diseases, type 2 diabetes mellitus, neurocognitive disorders, and general mortality. The role of vitamin D in gastric homeostasis is crucial and unexplored. This review attempts to elucidate the central role of vitamin D in preserving and maintaining the overall health and homeostasis of the stomach tissue.

Probiotics Upregulate Trefoil Factors and Downregulate Pepsinogen in the Mouse Stomach.

Probiotics are used in the management of some gastrointestinal diseases. However, little is known about their effects on normal gastric epithelial biology. The aim of this study was to explore how the probiotic mixture VSL#3 affects gastric cell lineages in mice with a special focus on protective and aggressive factors. Weight-matching littermate male mice (n = 14) were divided into treated and control pairs. The treated mice received VSL#3 (5 mg/day/mouse) by gastric gavage for 10 days. Control mice received only the vehicle. Food consumption and bodyweight were monitored. All mice were injected intraperitoneally with bromodeoxyuridine (120 mg/Kg bodyweight) two hours before sacrificed to label S-phase cells. Stomach tissues were processed for lectin- and immunohistochemical examination. ImageJ software was used to quantify immunolabeled gastric epithelial cells. Real-time quantitative polymerase chain reaction was used to provide relative changes in expression of gastric cell lineages specific genes. Results revealed that treated mice acquired (i) increased production of mucus, trefoil factor (TFF) 1 and TFF2, (ii) decreased production of pepsinogen, and (iii) increased ghrelin-secreting cells. No significant changes were observed in bodyweight, food consumption, cell proliferation, or parietal cells. Therefore, VSL#3 administration amplifies specific cell types specialized in the protection of the gastric epithelium.

Gastrin induces parathyroid hormone-like hormone expression in gastric parietal cells.

Parietal cells play a fundamental role in stomach maintenance, not only by creating a pathogen-free environment through the production of gastric acid, but also by secreting growth factors important for homeostasis of the gastric epithelium. The gastrointestinal hormone gastrin is known to be a central regulator of both parietal cell function and gastric epithelial cell proliferation and differentiation. Our previous gene expression profiling studies of mouse stomach identified parathyroid hormone-like hormone (PTHLH) as a potential gastrin-regulated gastric growth factor. Although PTHLH is commonly overexpressed in gastric tumors, its normal expression, function, and regulation in the stomach are poorly understood. In this study we used pharmacologic and genetic mouse models as well as human gastric cancer cell lines to determine the cellular localization and regulation of this growth factor by the hormone gastrin. Analysis of PthlhLacZ/+ knock-in reporter mice localized Pthlh expression to parietal cells in the gastric corpus. Regulation by gastrin was demonstrated by increased Pthlh mRNA abundance after acute gastrin treatment in wild-type mice and reduced expression in gastrin-deficient mice. PTHLH transcripts were also observed in normal human stomach as well as in human gastric cancer cell lines. Gastrin treatment of AGS-E gastric cancer cells induced a rapid and robust increase in numerous PTHLH mRNA isoforms. This induction was largely due to increased transcriptional initiation, although analysis of mRNA half-life showed that gastrin treatment also extended the half-life of PTHLH mRNA, suggesting that gastrin regulates expression by both transcriptional and posttranscriptional mechanisms.NEW & NOTEWORTHY We show that the growth factor parathyroid hormone-like hormone (PTHLH) is expressed in acid-secreting parietal cells of the mouse stomach. We define the specific PTHLH mRNA isoforms expressed in human stomach and in human gastric cancer cell lines and show that gastrin induces PTHLH expression via transcription activation and mRNA stabilization. Our findings suggest that PTHLH is a gastrin-regulated growth factor that might contribute to gastric epithelial cell homeostasis.

Seven-Month Vitamin D Deficiency Inhibits Gastric Epithelial Cell Proliferation, Stimulates Acid Secretion, and Differentially Alters Cell Lineages in the Gastric Glands.

Vitamin D (VD) deficiency can result from insufficiency of either light exposure or VD intake. We investigated the biological effects of VD deficiency for 7 months on the mouse gastric glands. Varying degrees of VD deficiency were induced in C57BL/6 mice by keeping them on standard diet with constant-dark conditions (SDD) or VD deficient diet with constant-dark conditions (VDD). Samples of serum, glandular stomach, and gastric contents were collected for LCMS/MS, RT-PCR, immunohistochemistry, and acid content measurements. Both SDD and VDD mice had a significant decline in 25OHVD metabolite, gastric epithelial cell proliferation, and mucin 6 gene expression. These effects were enhanced with the severity of VD deficiency from SDD to VDD. Besides and compared to the control group, SDD mice only displayed a significant increase in the number of zymogenic cells (p ≤ 0.0001) and high expression of the adiponectin (p ≤ 0.05), gastrin (p ≤ 0.0001), mucin 5AC (*** p ≤ 0.001) and the Cyclin-dependent kinase inhibitor 1A (**** p ≤ 0.0001). These phenotypes were unique to SDD gastric samples and not seen in the VDD or control groups. This study suggests that the body reacts differently to diverse VD deficiency sources, light or diet.

Overgrowth of Squamocolumnar Junction and Dysregulation of Stem Cell Lineages in the Stomach of Vitamin A-Deficient Mice.

Junctional epithelia are common sites for pathological transformations. In mice, the stratified epithelium of the forestomach joins the simple glandular epithelium of the cardia at the limiting ridge. We previously demonstrated the expression of vitamin A receptors in the gastric stem/progenitor cells and their progeny and found that excess retinoic acid enhances cellular dynamics of gastric epithelium. This study examines how deficiency of vitamin A would alter gastric epithelial stem cell lineages. Three-week-old mice of both genders were weaned and fed with a vitamin A deficient (VAD) diet for 4 or 8 months. Sex- and weight-matched littermate mice received a standard (control) diet. To label S-phase cells, all mice received a single intraperitoneal injection of 5-bromo-2-deoxyuridine before being euthanized. Stomach tissues were processed for microscopic examination and protein analysis to investigate stem cell lineages using different stains, lectins, or antibodies. The Student's t-test was used to compare quantified data showing differences between control and VAD groups. Eight-month-vitamin-A deficiency caused enlarged forestomach and overgrowth of the squamocolumnar junction with metaplastic and dysplastic cardiac glands, formation of intramucosal cysts, loss of surface mucosal integrity, increased amount of luminal surface mucus, and upregulation of trefoil factor 1 and H+,K+-ATPase. These changes were associated with decreased cell proliferation and upregulation of p63. In conclusion, vitamin A is necessary for maintaining gastric epithelial integrity and its deficiency predisposes the mouse stomach to precancerous lesions.

Hip1r is expressed in gastric parietal cells and is required for tubulovesicle formation and cell survival in mice.

Huntingtin interacting protein 1 related (Hip1r) is an F-actin- and clathrin-binding protein involved in vesicular trafficking. In this study, we demonstrate that Hip1r is abundantly expressed in the gastric parietal cell, predominantly localizing with F-actin to canalicular membranes. Hip1r may provide a critical function in vivo, as demonstrated by extensive changes to parietal cells and the gastric epithelium in Hip1r-deficient mice. Electron microscopy revealed abnormal apical canalicular membranes and loss of tubulovesicles in mutant parietal cells, suggesting that Hip1r is necessary for the normal trafficking of these secretory membranes. Accordingly, acid secretory dynamics were altered in mutant parietal cells, with enhanced activation and acid trapping, as measured in isolated gastric glands. At the whole-organ level, gastric acidity was reduced in Hip1r-deficient mice, and the gastric mucosa was grossly transformed, with fewer parietal cells due to enhanced apoptotic cell death and glandular hypertrophy associated with cellular transformation. Hip1r-deficient mice had increased expression of the gastric growth factor gastrin, and mice mutant for both gastrin and Hip1r exhibited normalization of both proliferation and gland height. Taken together, these studies demonstrate that Hip1r plays a significant role in gastric physiology, mucosal architecture, and secretory membrane dynamics in parietal cells.

Modulation of Stem Cell Progeny by Probiotics during Regeneration of Gastric Mucosal Erosions.

Patients with gastric mucosal erosions are predisposed to chronic gastritis, ulcer or even cancer. The repair of mucosal erosions involves several events including proliferation of gastric epithelial stem cells. The aim of this study was to investigate the effects of the probiotic mixture of De Simone Formulation on gastric epithelial stem cell lineages in mouse models of gastric mucosal erosions. Gastric erosions were induced by a single oral gavage of 80% ethanol containing 15 mg/mL acetylsalicylic acid (5 mL/kg) following a daily dose of probiotic mixture (5 mg/day/mouse) for 10 days. In another protocol, erosions were induced by a daily gavage of acetylsalicylic acid (400 mg/kg/day/mouse) for 5 days before or after daily administration of probiotic mixture for 5 days. Control mice received water gavage for 10 days. All mice were injected with bromodeoxyuridine two hours before sacrifice to label S-phase cells. The stomachs of all mice were processed for histological examination, lectin binding, and immunohistochemical analysis. The results reveal that mice that received probiotics before or after the induction of erosion showed a decrease in erosion index with an increase in gastric epithelial stem/progenitor cell proliferation and enhanced production of mucus, trefoil factors, and ghrelin by mucous and enteroendocrine cell lineages. These mice also showed restoration of the amount of H+,K+-ATPase and pepsinogen involved in the production of the harsh acidic environment by parietal and chief cell lineages. In conclusion, this study demonstrates the beneficial effects of probiotics against gastric mucosal erosion and highlights the involvement and modulation of proliferative stem cells and their multiple glandular epithelial cell lineages.

Vitamin D Is Necessary for Murine Gastric Epithelial Homeostasis.

Unlike other organs, the importance of VD in a normal stomach is unknown. This study focuses on understanding the physiological role of vitamin D in gastric epithelial homeostasis. C57BL/6J mice were divided into three groups that were either fed a standard diet and kept in normal light/dark cycles (SDL), fed a standard diet but kept in the dark (SDD) or fed a vitamin D-deficient diet and kept in the dark (VDD). After 3 months, sera were collected to measure vitamin D levels by LC-MS/MS, gastric tissues were collected for immunohistochemical and gene expression analyses and gastric contents were collected to measure acid levels. The VDD group showed a significant decrease in the acid-secreting parietal cell-specific genes Atp4a and Atp4b when compared with the controls. This reduction was associated with an increased expression of an antral gastrin hormone. VDD gastric tissues also showed a high proliferation rate compared with SDL and SDD using an anti-BrdU antibody. This study indicates the requirement for normal vitamin D levels for proper parietal cell functions.

Application of a new vitamin D blood test on the Emirati population.

Research shows that immunoassay techniques are not the best choice for the estimation of vitamin D in human blood samples. The main reasons are that some immunoassays are not able to distinguish between 25-OHD3 and 25-OHD2 vitamin D metabolites. Furthermore, immunoassays cannot differentiate between 25OHD and inactive epimers of vitamin D. Vitamin D epimers and isobars have been known to overlap with the 25OHD signals and give false positives when tested. Liquid chromatography tandem-mass spectrometry (LC-MS/MS) can differentiate between 25OHD3 and 25OHD2. Separating epimers and isobars (which have the same molecular weight) from vitamin D is achieved through chromatographic separation from actual 25OHD peaks, although this could also cause inaccuracies in vitamin D measurements. The main aim of this study was to develop and validate an improved LC-MS/MS method (using a Shimadzu 8060 system) that could accurately detect and quantitate up to 10 different metabolites of vitamin D, as well as differentiate the epimers and isobars. The secondary aim was to apply the developed LC-MS/MS method for the accurate measurement of blood vitamin D levels in the Emirati population. The Shimadzu 8060 system was run using positive ion electrospray ionization (ESI) in Dynamic Multiple Reaction Monitoring (DMRM) mode for quantification. The method involved blood sample collection from 80 Emirati volunteers, followed by serum extraction and liquid-liquid extraction. The chromatography column used for the analysis was an Ascentis Express F5. Precursor and product ions were detected using a Shimadzu 8060 LC-MS/MS system, and 10 metabolites of vitamin D were detected and quantified, including epimers and isobars. The method validation showed good sensitivity, recovery, linearity, precision, specificity, and accuracy. Furthermore, the data showed that vitamin D epimer 3-epi-25OHD and isobar 7-α-hydroxy-4-cholesten-3-one (7αC4) accounted for a significant portion of vitamin D results in the Emirati population. We report a more reliable, reproducible, and robust LC-MS/MS method for the accurate detection of 25OHD (vitamin D) in the Emirati population. The method has the capacity to detect and separate 10 metabolites of vitamin D as well as separate 25OHD from co-eluting epimers and isobars. The method has also been successfully implemented in gauging vitamin D deficiency in the Emirati population. Thus, this improved LC-MS/MS method could prove very useful in accurately estimating the levels of vitamin D in the Emirati population and in further clinical studies.

Potential role of probiotics in the management of gastric ulcer.

Gastric ulcer is one of the most common chronic gastrointestinal diseases characterized by a significant defect in the mucosal barrier. Helicobacter pylori (H. pylori) infection and the frequent long-term use of non-steroidal anti-inflammatory drugs are major factors involved in gastric ulcer development. Acid inhibitors and antibiotics are commonly used to treat gastric ulcer. However, in the last few decades, the accumulating evidence for resistance to antibiotics and the side effects of antibiotics and acid inhibitors have drawn attention to the possible use of probiotics in the prevention and treatment of gastric ulcer. Probiotics are live microorganisms that when administered in adequate amounts confer health benefits on the host. Currently, the available experimental and clinical studies indicate that probiotics are promising for future applications in the management of gastric ulcers. This review aims to provide an overview of the general health benefits of probiotics on various systemic and gastrointestinal disorders with a special focus on gastric ulcer and the involved cellular and molecular mechanisms: i) Protection of gastric mucosal barrier; ii) upregulation of prostaglandins, mucus, growth factors and anti-inflammatory cytokines; iii) increased cell proliferation to apoptosis ratio; and iv) induction of angiogenesis. Finally, some of the available data on the possible use of probiotics in H. pylori eradication are discussed.