BackgroundRecent studies demonstrated that Aquamin®, a calcium-, magnesium-rich, multi-mineral natural product, improves barrier structure and function in colonoids obtained from the tissue of healthy subjects. The goal of the present study was to determine if the colonic barrier could be improved in tissue from subjects with ulcerative colitis (UC).MethodsColonoid cultures were established with colon biopsies from 9 individuals with UC. The colonoids were then incubated for a 2-week period under control conditions (in culture medium with a final calcium concentration of 0.25 mM) or in the same medium supplemented with Aquamin® to provide 1.5 – 4.5 mM calcium. Effects on differentiation and barrier protein expression were determined using several approaches: phase-contrast and scanning electron microscopy, quantitative histology and immunohistology, mass spectrometry-based proteome assessment and transmission electron microscopy.ResultsAlthough there were no gross changes in colonoid appearance, there was an increase in lumen diameter and wall thickness on histology and greater expression of cytokeratin 20 (CK20) along with reduced expression of Ki67 by quantitative immunohistology observed with intervention. In parallel, upregulation of several differentiation-related proteins was seen in a proteomic screen with the intervention. Aquamin®-treated colonoids demonstrated a modest up-regulation of tight junctional proteins but stronger induction of adherens junction and desmosomal proteins. Increased desmosomes were seen at the ultrastructural level. Proteomic analysis demonstrated increased expression of several basement membrane proteins and hemidesmosomal components. Proteins expressed at the apical surface (mucins and trefoils) were also increased as were several additional proteins with anti-microbial activity or that modulate inflammation. Finally, several transporter proteins that affect electrolyte balance (and, thereby affect water resorption) were increased. At the same time, growth and cell cycle regulatory proteins (Ki67, nucleophosmin, and stathmin) were significantly down-regulated. Laminin interactions, matrix formation and extracellular matrix organization were the top three up-regulated pathways with the intervention.ConclusionA majority of individuals including patients with UC do not reach the recommended daily intake for calcium and other minerals. To the extent that such deficiencies might contribute to the weakening of the colonic barrier, the findings employing UC tissue-derived colonoids here suggest that adequate mineral intake might improve the colonic barrier.

The overall goal of this study was to determine whether Aquamin®, a calcium-, magnesium-, trace element-rich, red algae-derived natural product, would alter the expression of proteins involved in growth-regulation and differentiation in colon. Thirty healthy human subjects (at risk for colorectal cancer) were enrolled in a three-arm, 90-day interventional trial. Aquamin® was compared to calcium alone and placebo. Before and after the interventional period, colonic biopsies were obtained. Biopsies were evaluated by immunohistology for expression of Ki67 (proliferation marker) and for CK20 and p21 (differentiation markers). Tandem mass tag-mass spectrometry-based detection was used to assess levels of multiple proteins. As compared to placebo or calcium, Aquamin® reduced the level of Ki67 expression and slightly increased CK20 expression. Increased p21 expression was observed with both calcium and Aquamin®. In proteomic screen, Aquamin® treatment resulted in many more proteins being upregulated (including pro-apoptotic, cytokeratins, cell–cell adhesion molecules, and components of the basement membrane) or downregulated (proliferation and nucleic acid metabolism) than placebo. Calcium alone also altered the expression of many of the same proteins but not to the same extent as Aquamin®. We conclude that daily Aquamin® ingestion alters protein expression profile in the colon that could be beneficial to colonic health.

Male MS-NASH mice were maintained on a high-fat diet for 16 weeks with and without red algae-derived minerals. Obeticholic acid (OCA) was used as a comparator in the same strain and diet. C57BL/6 mice maintained on a standard (low-fat) rodent chow diet were used as a control. At the end of the in-life portion of the study, body weight, liver weight, liver enzyme levels and liver histology were assessed. Samples obtained from individual livers were subjected to Tandem Mass Tag labeling / mass spectroscopy for protein profile determination. As compared to mice maintained on the low-fat diet, all high-fat-fed mice had increased whole-body and liver weight, increased liver enzyme (aminotransferases) levels and widespread steatosis / ballooning hepatocyte degeneration. Histological evidence for liver inflammation and collagen deposition was also present, but changes were to a lesser extent. A moderate reduction in ballooning degeneration and collagen deposition was observed with mineral supplementation. Control mice on the high-fat diet alone demonstrated multiple protein changes associated with dysregulated fat and carbohydrate metabolism, lipotoxicity and oxidative stress. Cholesterol metabolism and bile acid formation were especially sensitive to diet. In mice receiving multi-mineral supplementation along with the high-fat diet, there was reduced liver toxicity as evidenced by a decrease in levels of several cytochrome P450 enzymes and other oxidant-generating moieties. Additionally, elevated expression of several keratins was also detected in mineral-supplemented mice. The protein changes observed with mineral supplementation were not seen with OCA. Our previous studies have shown that mice maintained on a high-fat diet for up to 18 months develop end-stage liver injury including hepatocellular carcinoma. Mineral-supplemented mice were substantially protected against tumor formation and other end-state consequences of high-fat feeding. The present study identifies early (16-week) protein changes occurring in the livers of the high-fat diet-fed mice, and how the expression of these proteins is influenced by mineral supplementation. These findings help elucidate early protein changes that contribute to end-stage liver injury and potential mechanisms by which dietary minerals may mitigate such damage.

Background: Recent studies demonstrated that Aquamin®, a calcium-, magnesium-, and multiple trace element-rich natural product, improves barrier structure and function in colonoids obtained from the tissue of healthy subjects. The goal of the present study was to determine if the colonic barrier could be improved in tissue from subjects with ulcerative colitis (UC). Methods: Colonoid cultures were established with tissue from 9 individuals with UC. The colonoids were then incubated for a 2-week period under control conditions (i.e., in culture medium with a final calcium concentration of 0.25 mM) or in the same medium supplemented with Aquamin® to provide 1.5 − 4.5 mM calcium. Effects on differentiation and barrier protein expression were determined using several approaches: phase-contrast & scanning electron microscopy, quantitative histology & immunohistology, mass spectrometry-based proteome assessment and transmission electron microscopy. Results: Aquamin®-treated colonoids demonstrated a modest up-regulation of tight junctional proteins but stronger induction of adherens junction proteins and desmosomal proteins. Increased desmosomes were seen at the ultrastructural level. Proteomic analysis also demonstrated increased expression of basement membrane proteins and hemidesmosomal components. Proteins expressed at the apical surface (mucins and trefoils) were also increased as were several additional proteins with anti-microbial activity or that modulate inflammation. Conclusion: A majority of individuals including patients with UC do not reach the recommended daily intake for calcium and other minerals. The findings presented here suggest that adequate mineral intake might improve the colonic barrier.

The overall goal of this study was to determine if Aquamin®, a calcium- and magnesium-rich natural product, would alter the expression of proteins involved in growth-regulation, differentiation and barrier formation in the colon. Thirty healthy human subjects were enrolled in a three-arm, 90-day interventional trial in which Aquamin® (provided daily to deliver 800-mg of calcium per day) was compared to calcium alone and placebo. Before and after the 90-day interventional period, colonic biopsies were obtained. Biopsies were evaluated by immunohistology for expression of Ki67 (a proliferation marker) and for CK20 and p21 (differentiation markers). Tandem mass tag-mass spectrometry-based detection was used to assess levels of multiple proteins. As compared to placebo or calcium, Aquamin® reduced the level of Ki67 expression (20%). Neither intervention altered CK20 expression, while a trend toward increased p21 was observed with calcium and Aquamin® (117% and 99% respectively). In the proteomic screen, Aquamin® treatment resulted in many more proteins being upregulated or downregulated (1.5 fold-change with ≤2% false-discovery rate) than placebo. Included among the upregulated proteins were cytokeratins, cell-cell adhesion molecules and components of the basement membrane. Many of the downregulated proteins were those involved in proliferation and nucleic acid metabolism. Calcium alone also altered the expression of many of the same proteins but not to the same extent as Aquamin®. We conclude that daily Aquamin® ingestion alters protein expression profile in the colon that could be beneficial to colonic health. These data warrant additional studies with a larger sample size to validate these findings.Prevention RelevanceA multi-mineral approach reduced proliferation and induced differentiation in ex vivo settings and has been shown to decrease colon polyp incidence in mouse (polyp-prevention) studies. The findings from a 90-day trial in human subjects (presented here) demonstrated improved biomarker-modulation efficacy, warranting to conduct the polyp-prevention trial in at-risk human subjects.