Colonic Absorption Research Articles

Drug Disposition in the Lower Gastrointestinal Tract: Targeting and Monitoring.

The increasing prevalence of colonic diseases calls for a better understanding of the various colonic drug absorption barriers of colon-targeted formulations, and for reliable in vitro tools that accurately predict local drug disposition. In vivo relevant incubation conditions have been shown to better capture the composition of the limited colonic fluid and have resulted in relevant degradation and dissolution kinetics of drugs and formulations. Furthermore, drug hurdles such as efflux transporters and metabolising enzymes, and the presence of mucus and microbiome are slowly integrated into drug stability- and permeation assays. Traditionally, the well characterized Caco-2 cell line and the Ussing chamber technique are used to assess the absorption characteristics of small drug molecules. Recently, various stem cell-derived intestinal systems have emerged, closely mimicking epithelial physiology. Models that can assess microbiome-mediated drug metabolism or enable coculturing of gut microbiome with epithelial cells are also increasingly explored. Here we provide a comprehensive overview of the colonic physiology in relation to drug absorption, and review colon-targeting formulation strategies and in vitro tools to characterize colonic drug disposition.

Sustained-release hot melt extrudates of the weak acid TMP-001: A case study using PBB modelling

Over the last 30 years, hot melt extrusion has become a leading technology in the manufacture of amorphous drug delivery systems. Mostly applied as an ‘enabling formulation’ for poorly soluble compounds, application in the design of sustained-release formulations increasingly attracts the attention of the pharmaceutical industry.The drug candidate TMP-001 is currently under evaluation for the early treatment of Multiple Sclerosis. Although this weak acid falls into class II of the Biopharmaceutics Classification System, the compound exhibits high solubility in the upper intestine resulting in high peroral bioavailability. In the present studies, four different formulation prototypes varying in their sustained-release behavior were developed, using L-arginine as a pore-forming agent in concentrations ranging between 0 and 20%. Initially, biorelevant release testing was applied to assess the dissolution behavior of the prototypes. For these formulations, a total drug release of 44.7%, 64.6%, 75%, and 90.5% was achieved in FaSSIF-v2 after 24 h. Two candidates were selected for further characterization considering the crystal structure and the physical stability of the amorphous state of TMP-001 in the formulations together with the release behavior in Level II biorelevant media. Our findings indicate L-arginine as a valuable excipient in the formulation of hot melt extrudates, as its presence led to a considerable stabilization of the amorphous state and favorably impacted the milling process and release behavior of TMP-001. To properly evaluate the proposed formulations and the importance of colonic dissolution and absorption on the overall bioavailability, a physiologically-based biopharmaceutics model was used.

Increased colonic K+ excretion through inhibition of the H,K-ATPase type 2 helps reduce plasma K+ level in a murine model of nephronic reduction

Hyperkalemia is frequently observed in patients at the end-stage of chronic kidney disease (CKD), and has possible harmful consequences on cardiac function. Many strategies are currently used to manage hyperkalemia, one consisting of increasing fecal K+ excretion through the administration of cation-exchange resins. In this study, we explored another more specific method of increasing intestinal K+ secretion by inhibiting the H,K-ATPase type 2 (HKA2), which is the main colonic K+ reabsorptive pathway. We hypothetised that the absence of this pump could impede the increase of plasma K+ levels following nephronic reduction (N5/6) by favoring fecal K+ secretion. In N5/6 WT and HKA2KO mice under normal K+ intake, the plasma K+ level remained within the normal range, however, a load of K+ induced strong hyperkalemia in N5/6 WT mice (9.1 ± 0.5 mM), which was significantly less pronounced in N5/6 HKA2KO mice (7.9 ± 0.4 mM, p < 0.01). This was correlated to a higher capacity of HKA2KO mice to excrete K+ in their feces. The absence of HKA2 also increased fecal Na+ excretion by inhibiting its colonic ENaC-dependent absorption. We also showed that angiotensin-converting-enzyme inhibitor like enalapril, used to treat hypertension during CKD, induced a less severe hyperkalemia in N5/6 HKA2KO than in N5/6 WT mice. This study therefore provides the proof of concept that the targeted inhibition of HKA2 could be a specific therapeutic maneuver to reduce plasma K+ levels in CKD patients.

Magnetic resonance colonography with intestine-absorbable nanoparticle contrast agents in evaluation of colorectal inflammation

ObjectivesTo develop a nanoparticle-based MRI protocol based on transrectal administration of intestine-absorbable nanoparticle contrast agents to evaluate ulcerative colitis (UC).MethodsSolid lipid nanoparticles (SLNs) were synthesized by loading gadolinium diethylenetriaminepentaacetic acid (Gd-DTPA) and octadecylamine-fluorescein isothiocyanate to produce Gd-FITC-SLNs as T1 contrast agents. Twenty mice with acute UC were divided into four groups: enema with Gd-FITC-SLNs, intravenous injection of Gd-FITC-SLNs, enema with Gd-DTPA, and intravenous injection of Gd-DTPA. Five mice with chronic UC and five mice without UC underwent enema with Gd-FITC-SLNs. Axial T1- and T2-weighted MR images were obtained before and 20, 40, 60, 80,100, and 120 min after enema or intravenous injection of the contrast agent. The signal-to-noise ratios (SNRs) of the colorectal wall were measured in both groups. The MRI findings were correlated with subsequent histological confirmation.ResultsAt 20 min after enema with Gd-FITC-SLNs, MRI showed the following contrast enhancement pattern: acute UC > normal intestinal wall > chronic UC. A continuous enhancement effect was observed in mice with acute UC, whereas a slight continuous enhancement of the colorectal wall was observed in mice with chronic UC. The normal intestinal wall rapidly metabolized the contrast agent, and the enhancement decreased on sequential scans. There was no significant difference between the SNRs of the intestinal wall at 20 min after intravenous Gd-DTPA and transrectal Gd-FITC-SLN administration.ConclusionsEnema with Gd-FITC-SLNs may be helpful for the diagnosis and differential diagnosis of acute and chronic UC and can confer the same or better results than with intravenous Gd-DTPA.Key Points• Enema with Gd-FITC-SLNs may be helpful for the diagnosis and differential diagnosis of acute and chronic UC.• Enema with Gd-FITC-SLNs can achieve the same or better result than that with intravenous Gd-DTPA.• SLN-based MR colonography enhances the colorectal wall inflammation, based on the colonic absorption of the nanoparticle contrast agents.

Increased intestinal phosphate absorption, an often-overlooked effect of vitamin D.

Increased intestinal phosphate absorption, an often-overlooked effect of vitamin D.

Bioavailability and nutrikinetics of rosemary tea phenolic compounds in humans

Rosmarinus officinalis L. is a widespread aromatic plant commonly consumed as a tea in traditional cuisine and in folk medicine to treat various illnesses due to its therapeutic properties. To the best of our knowledge, there are no reports on the bioavailability and metabolism of R. officinalis tea polyphenols in humans. This study was aimed at assessing the bioavailability and nutrikinetics of R. officinalis phenolic compounds in healthy humans for the first time. Forty-eight compounds were identified in plasma and urine. Few un-metabolized compounds were detected since rosemary polyphenols were extensively metabolized into phase II conjugates, with rapid appearance and clearance in plasma, pointing to small intestinal absorption. Phase II derivatives of caffeic acid showed kinetics compatible with both intestinal and colonic hydrolysis of rosmarinic acid yielding free caffeic and 3,4-dihydroxyphenyl-lactic acids, which were absorbed and metabolized into phase II derivatives. These metabolites, along with reduced forms of caffeic acid and their phase II metabolites, and those of hydroxyphenylpropionic, hydroxylphenylacetic, benzoic and hippuric acids, highlight the importance of colonic absorption. Total urinary excretion of the phenols added up to 235 µmol, corresponding to 22.3% of the ingested amount (1055 µM). In conclusion, rosemary tea polyphenols are partially bioavailable and extensively metabolized, mainly by the colonic microbiota.

A novel delivery system for enhancing bioavailability of S-adenosyl-l-methionine: Pectin nanoparticles-in-microparticles and their in vitro - in vivo evaluation'

Nanoparticles-in-microparticles (NIMs) are novel drug delivery systems, which combine the benefits of nano-, and micro-sized drug carriers within one system to improve the bioavailability of drugs or provide site specific drug delivery. The goal of this work was to study the in vitro and in vivo characteristics of a new pectin nanoparticle based NIM formulation for colon targeted delivery of S-adenosyl-l-methionine (SAMe). The effects of different formulation parameters on physicochemical characteristics of pectin nanoparticles prepared by ionic-gelation method were estimated by a factorial design. The studied formulation variables were different amounts of drug, pectin and crosslinking agent. NIMs were prepared using fluidized bed technology by coating the optimum nanoparticle formulation (particle size 301.5 ± 20.3 nm, zeta potential: −16.3 ± 1.7 mV, entrapment efficiency: 74.80 ± 3.75%, product yield: 69.61%) on inert pellets via subcoating and pH- sensitive functional coating materials. NIMs were characterized by particle size, surface charge, morphology, entrapment efficiency, DSC, XRPD, in vitro drug release in the presence and absence of enzymes (pepsin and pectinase) in simulated gastric and colonic fluids. The in vitro drug release studies suggested retarded drug release, which was interpreted as an advantage for improving oral bioavailability of SAMe via colonic absorption. Comparative bioavailability of SAMe was determined after oral administration of pure SAMe solution, SAMe loaded pectin nanoparticles and NIMs to Wistar rats. Although inter-subject variability was observed, the relative bioavailability of SAMe (260%) increased via oral NIMs administration. This outcome should be supported with further bioavailability studies in larger groups. Overall, this study suggests that developed fluidized bed coating technique can be used to construct solid microparticular systems for colonic delivery of nanoparticles and overcome the difficulties in processing nanoparticles.

Enhanced oral delivery of insulin via the colon-targeted nanocomposite system of organoclay/glycol chitosan/Eudragit\xaeS100

This study aimed to develop a ternary nanocomposite system of organoclay, glycol-chitosan, and Eudragit®S100 as an effective colon targeted drug delivery carrier to enhance the oral absorption of insulin. A nanocomplex of insulin and aminoclay was prepared via spontaneous co-assembly, which was then coated with glycol-chitosan and Eudragit S®100 (EGAC-Ins). The double coated nanocomplex, EGAC-Ins demonstrated a high entrapment efficiency of greater than 90% and a pH-dependent drug release. The conformational stability of insulin entrapped in EGAC-Ins was effectively maintained in the presence of proteolytic enzymes. When compared to a free insulin solution, EGAC-Ins enhanced drug permeability by approximately sevenfold in Caco-2 cells and enhanced colonic drug absorption in rats. Accordingly, oral EGAC-Ins significantly reduced blood glucose levels in diabetic rats while the hypoglycemic effect of an oral insulin solution was negligible. In conclusion, EGAC-Ins should be a promising colonic delivery system for improving the oral absorption of insulin.

Parallel intermediate conductance K+ and Cl- channel activity mediates electroneutral K+ exit across basolateral membranes in rat distal colon.

Transepithelial K+ absorption requires apical K+ uptake and basolateral K+ exit. In the colon, apical H+-K+-ATPase mediates cellular K+ uptake, and it has been suggested that electroneutral basolateral K+ exit reflects K+-Cl- cotransporter-1 (KCC1) operating in parallel with K+ and Cl- channels. The present study was designed to identify basolateral transporter(s) responsible for K+ exit in rat distal colon. Active K+ absorption was determined by measuring 86Rb+ (K+ surrogate) fluxes across colonic epithelia under voltage-clamp conditions. With zero Cl- in the mucosal solution, net K+ absorption was reduced by 38%, indicating that K+ absorption was partially Cl--dependent. Serosal addition of DIOA (KCC1 inhibitor) or Ba2+ (nonspecific K+ channel blocker) inhibited net K+ absorption by 21% or 61%, respectively, suggesting that both KCC1 and K+ channels contribute to basolateral K+ exit. Clotrimazole and TRAM34 (IK channel blockers) added serosally inhibited net K+ absorption, pointing to the involvement of IK channels in basolateral K+ exit. GaTx2 (CLC2 blocker) added serosally also inhibited net K+ absorption, suggesting that CLC2-mediated Cl- exit accompanies IK channel-mediated K+ exit across the basolateral membrane. Net K+ absorption was not inhibited by serosal addition of either IbTX (BK channel blocker), apamin (SK channel blocker), chromanol 293B (KV7 channel blocker), or CFTRinh172 (CFTR blocker). Immunofluorescence studies confirmed basolateral membrane colocalization of CLC2-like proteins and Na+-K+-ATPase α-subunits. We conclude that active K+ absorption in rat distal colon involves electroneutral basolateral K+ exit, which may reflect IK and CLC2 channels operating in parallel.NEW & NOTEWORTHY This study demonstrates that during active electroneutral K+ absorption in rat distal colon, K+ exit across the basolateral membrane mainly reflects intermediate conductance K+ channels operating in conjunction with chloride channel 2, with a smaller, but significant, contribution from K+-Cl- cotransporter-1 (KCC1) activity.

Regional Intestinal Drug Permeability and Effects of Permeation Enhancers in Rat.

Sufficient colonic absorption is necessary for all systemically acting drugs in dosage forms that release the drug in the large intestine. Preclinically, colonic absorption is often investigated using the rat single-pass intestinal perfusion model. This model can determine intestinal permeability based on luminal drug disappearance, as well as the effect of permeation enhancers on drug permeability. However, it is uncertain how accurate the rat single-pass intestinal perfusion model predicts regional intestinal permeability and absorption in human. There is also a shortage of systematic in vivo investigations of the direct effect of permeation enhancers in the small and large intestine. In this rat single-pass intestinal perfusion study, the jejunal and colonic permeability of two low permeability drugs (atenolol and enalaprilat) and two high-permeability ones (ketoprofen and metoprolol) was determined based on plasma appearance. These values were compared to already available corresponding human data from a study conducted in our lab. The colonic effect of four permeation enhancers—sodium dodecyl sulfate, chitosan, ethylenediaminetetraacetic acid (EDTA), and caprate—on drug permeability and transport of chromium EDTA (an established clinical marker for intestinal barrier integrity) was determined. There was no difference in jejunal and colonic permeability determined from plasma appearance data of any of the four model drugs. This questions the validity of the rat single-pass intestinal perfusion model for predicting human regional intestinal permeability. It was also shown that the effect of permeation enhancers on drug permeability in the colon was similar to previously reported data from the rat jejunum, whereas the transport of chromium EDTA was significantly higher (p < 0.05) in the colon than in jejunum. Therefore, the use of permeation enhancers for increasing colonic drug permeability has greater risks than potential medical rewards, as indicated by the higher permeation of chromium EDTA compared to the drugs.

Hydrophilic Poly(urethanes) Are an Effective Tool for Gastric Retention Independent of Drug Release Rate

Acyclovir is a poorly permeable, short half-life drug with poor colonic absorption, and current conventional controlled release formulations are unable to decrease the frequency of administration. We designed acyclovir dosage forms to be administered less frequently by being retained in the stomach and releasing drug over an extended duration. We developed a conventional modified-release matrix tablet to sustain the release of acyclovir and surrounded it with a hydrophilic poly(urethane) layer. When hydrated, the porous poly(urethane) swells to a size near or beyond that of the relaxed pylorus diameter and does not affect drug release rate. We demonstrated that the formulation is retained in the stomach for extended durations as it slowly releases drug, allowing for similar area under the curve but delayed tmax relative to a nongastroretentive control tablet. Unlike many other gastroretentive formulations, this dosage form design decouples drug release rate from gastric retention time, allowing them to be modulated independently. It also effectively retains in the stomach regardless of the prandial state, differentiating from other approaches. Our direct observation of excised rat stomachs allowed for a rigorous assessment of the impact of polymer swelling extent and the prandial state on both the dosage form integrity and retention time.

Impairment of electroneutral Na+transport and associated downregulation of NHE3 contributes to the development of diarrhea following in vivo challenge withBrachyspiraspp.

The effect of Brachyspira hyodysenteriae and Brachyspira hampsonii spirochetosis on Na+ transport was assessed in the colon to determine its contribution to diarrheal disease in pigs following experimental infection. Electrogenic and electroneutral Na+ absorption was assessed in Ussing chambers by radiolabeled 22Na flux and pharmacological inhibitory studies. Basal radiolabeled 22Na flux experiments revealed that mucosal-to-serosal flux (Jms) was significantly impaired in B. hyodysenteriae and B. hampsonii-diseased pigs. Inhibition of epithelial sodium channel via amiloride did not significantly reduce electrogenic short-circuit current (Isc) in the proximal, apex, and distal colonic segments of diseased pigs over control pigs, suggesting that a loss of electroneutral Na+ absorption is responsible for diarrheal development. These findings were further supported by significant downregulation of Na+/H+ exchanger (NHE1, NHE2, and NHE3) mRNA expression in the proximal, apex, and distal colonic segments paired with decreased protein expression of the critical NHE3 isoform. The decrease in NHE3 mRNA expression appears not to be attributed to the host's cytokine response as human IL-1α did not modify NHE3 mRNA expression in Caco-2 cells. However, a whole cell B. hampsonii lysate significantly downregulated NHE3 mRNA expression and significantly increased p38 phosphorylation in Caco-2 cells. Together these findings provide a likely mechanism for the spirochete-induced malabsorptive diarrhea, indicated by a decrease in electroneutral Na+ absorption in the porcine colon due to Brachyspira's ability to inhibit NHE3 transcription, resulting in diarrheal disease.NEW & NOTEWORTHY This research demonstrates that diarrheal disease caused by two infectious spirochete spp. is a result of impaired electroneutral Na+ absorption via Na+/H+ exchanger 3 (NHE3) in the porcine colon. Our findings suggest that the decrease in NHE3 mRNA and protein is not likely a result of the host's cytokine response. Rather, it appears that these two Brachyspira spp. directly inhibit the transcription and translation of NHE3, resulting in the development of diarrhea.

Absorptive transport of amino acids by the rat colon.

The capacity of the colon to absorb microbially produced amino acids (AAs) and the underlying mechanisms of AA transport are incompletely defined. We measured the profile of 16 fecal AAs along the rat ceco-colonic axis and compared unidirectional absorptive AA fluxes across mucosal tissues isolated from the rat jejunum, cecum, and proximal colon using an Ussing chamber approach, in conjunction with 1H-NMR and ultra-performance liquid chromatography-mass spectrometry chemical analyses. Passage of stool from cecum to midcolon was associated with segment-specific changes in fecal AA composition and a decrease in total AA content. Simultaneous measurement of up to 16 AA fluxes under native luminal conditions, with correction for endogenous AA release, demonstrated absorptive transfer of AAs across the cecum and proximal colon at rates comparable (30-80%) to those across the jejunum, with significant Na+-dependent and H+-stimulated components. Expression profiling of 30 major AA transporter genes by quantitative PCR revealed comparatively high levels of transcripts for 20 AA transporters in the cecum and/or colon, with the levels of 12 exceeding those in the small intestine. Our results suggest a more detailed model of major apical and basolateral AA transporters in rat colonocytes and provide evidence for a previously unappreciated transfer of AAs across the colonic epithelium that could link the prodigious metabolic capacities of the luminal microbiota, the colonocytes, and the body tissues.NEW & NOTEWORTHY This study provides evidence for a previously unappreciated transfer of microbially generated amino acids across the colonic epithelium under physiological conditions that could link the prodigious metabolic capacities of the luminal microbiota, the colonocytes, and the body tissues. The segment-specific expression of at least 20 amino acid transporter genes along the colon provides a detailed mechanistic basis for uniport, heteroexchange, Na+-cotransport, and H+-cotransport components of colonic amino acid absorption.

Increased DMT1 and FPN1 expression with enhanced iron absorption in ulcerative colitis human colon.

Iron deficiency anemia is a common complication of ulcerative colitis (UC) that can profoundly impact quality of life. Most iron absorption occurs in the duodenum via divalent metal transporter 1 (DMT1)-mediated uptake and ferroportin-1 (FPN1)-mediated export across the apical and basolateral membranes, respectively. However, the colon also contains iron transporters and can participate in iron absorption. Studies have shown increased duodenal DMT1 and FPN1 in patients with UC, but there is conflicting evidence about whether expression is altered in UC colon. We hypothesized that expression of colonic DMT1 and FPN1 will also increase to compensate for iron deficiency. Quantitative RT-PCR and Western blot analyses were performed on duodenal and colonic segmental (right colon, transverse colon, left colon, and rectum) biopsies obtained during colonoscopy. DMT1 mRNA and protein abundances in colonic segments were approximately equal to those in the duodenum, whereas colonic FPN1 mRNA and protein abundances of colonic segments were about one-quarter of those of the duodenum. DMT1 specific mRNA and protein abundances were increased twofold, whereas FPN1 mRNA and protein expressions were increased fivefold in UC distal colon. Immunofluorescence studies revealed enhanced expression of apical membrane- and basolateral membrane-localized DMT1 and FPN1 in UC human colon, respectively. Increased DMT1 expression was associated with enhanced 2-(3-carbamimidoylsulfanylmethyl-benzyl)-isothiourea (CISMBI, DMT1 specific inhibitor)-sensitive 59Fe uptake in UC human colon. We conclude from these results that patients with active UC have increased expression of colonic iron transporters and increased iron absorption, which may be targeted in the treatment of UC-related anemia.

Pharmacokinetic Drug-Drug Interactions Between Trospium Chloride and Ranitidine Substrates of Organic Cation Transporters in Healthy Human Subjects.

Trospium chloride, a muscarinic receptor blocker, is poorly absorbed with different rates from areas in the jejunum and the cecum/ascending colon. To evaluate whether organic cation transporter (OCT) 1, OCT2 and multidrug and toxin extrusion (MATE) 1 and MATE2-K are involved in pharmacokinetics, competitions with ranitidine, a probe inhibitor of the cation transporters, were evaluated in transfected HEK293 cells. Furthermore, a drug interaction study with trospium chloride after intravenous (2 mg) and oral dosing (30 mg) plus ranitidine (300 mg) was performed in 12 healthy subjects and evaluated by noncompartmental analysis and population pharmacokinetic modeling. Ranitidine inhibited OCT1, OCT2, MATE1, and MATE2-K with half maximal inhibitory concentration values of 186±25 µM, 482±105 µM, 134±37 µM, and 35±11 µM, respectively. In contrast to our hypothesis, coadministration of ranitidine did not significantly decrease oral absorption of trospium. Instead, renal clearance was lowered by ∼15% (530±99 vs 460±120 mL/min; P<.05). It is possible that ranitidine was not available in competitive concentrations at the major colonic absorption site, as the inhibitor is absorbed in the small intestine and undergoes degradation by microbiota. The renal effects apparently result from inhibition of MATE1 and/or MATE2-K by ranitidine as predicted by in vitro to in vivo extrapolation. However, all pharmacokinetic changes were not of clinical relevance for the drug with highly variable pharmacokinetics. Intravenous trospium significantly lowered mean absorption time and relative bioavailability of ranitidine, which was most likely caused by muscarinic receptor blocking effects on intestinal motility and water turnover.

Colonic Metabolism of Blueberry Polyphenols in Ovariectomized Rats Increases in a Dose-Response Fashion (OR34-06-19)

ObjectivesFruit and vegetable derived polyphenols are often touted for their health benefits and marketed as dietary supplements. Following consumption, polyphenols are extensively metabolized by gut microbiota to small molecular weight phenolics and absorbed in the colon. While these metabolites may drive observed health benefits, the pharmacokinetics of colonic absorption are not well characterized, especially at high dietary supplement doses. To address this, we investigated the dose-response of blueberry polyphenols and colonic metabolites produced over 48 h in a rat model. MethodsFive-month old, ovariectomized, Sprague-Dawley rats were fed a diet containing lyophilized, wild blueberries at 5% (w/w) for one week before being acutely dosed with purified wild blueberry phenolics at four doses (0, 60, 300, or 1100 mg total phenolics/kg bw) and 45Ca to measure calcium absorption. Blood and urine were collected for 48 h after dosing, with phenolic metabolites extracted, purified, and characterized via UPLC-MS/MS. ResultsTotal phenolic content of wild blueberries and the purified extract were 3.7% & 27% (w/w), respectively. Among individual phenolics, anthocyanins (1411 & 15,915 mg/100 g), chlorogenic acid (593 & 1916 mg/100 g), and quercetin species (275 & 2290 mg/100 g) were most prominent. After dosing, phenolic acids, including cinnamic and hippuric acids, were the most prominent microbial metabolites (530–2598 and 271–1263 nmol excreted, respectively); while only smaller amounts of anthocyanins (50–161 nmol excreted) were observed, including unmetabolized anthocyanins in the highest dose group. Most metabolites exhibited maximal blood and urine concentrations within 12 h of dosing, though some (e.g., hippuric acid) peaked up to 24 h after dosing. Finally, calcium absorption was significantly higher in the highest dose group as compared to the control group (p = 0.032). ConclusionsThese results indicate that colonic metabolites are absorbed later than their small intestinal counterparts, with variable peak absorption times. The increase in calcium absorption, coupled with the presence of unmetabolized anthocyanins in the highest dose group, suggests that high blueberry polyphenol doses may overwhelm normal metabolic pathways and potentially alter GI function. Funding SourcesNIH.

Impaired butyrate absorption in the proximal colon, low serum butyrate and diminished central effects of butyrate on blood pressure in spontaneously hypertensive rats.

Butyrate is a major gut microbiota-derived metabolite. Reduced butyrate-producing bacteria has been reported in the spontaneously hypertensive rat (SHR), a model of hypertension characterized by dysfunctional autonomic nervous system and gut dysbiosis. Here, we demonstrate a potential mechanism for butyrate in blood pressure regulation. High-performance liquid chromatography and liquid chromatography-mass spectrometry were performed to measure butyrate levels in feces and serum. Ussing chamber determined butyrate transport in colon ex vivo. Real-time PCR and immunohistochemistry evaluated expression of butyrate transporter, Slc5a8, in the colon. Mean arterial blood pressure was measured in catheterized anesthetized rats before and after a single butyrate intracerebroventricular injection. Activity of cardioregulatory brain regions was determined by functional magnetic resonance imaging to derive neural effects of butyrate. In the SHR, we demonstrated elevated butyrate levels in cecal content, but diminished butyrate levels in circulation, possibly due to reduced expression of Slc5a8 transporter in the colon. In addition, we observed lower expression levels of butyrate-sensing receptors in the hypothalamus of SHR, likely leading to the reduced effects of centrally administered butyrate on blood pressure in the SHR. Functional magnetic resonance imaging revealed reduced activation of cardioregulatory brain regions following central administration of butyrate in the SHR compared to control. We demonstrated a reduced availability of serum butyrate in the SHR, possibly due to diminished colonic absorption. Reduced expression of butyrate-sensing receptors in the SHR hypothalamus may explain the reduced central responsiveness to butyrate, indicating microbial butyrate may play a role in blood pressure regulation.

Simultaneous expression of ClopHensor and SLC26A3 reveals the nature of endogenous oxalate transport in CHO cells.

ABSTRACTClopHensor, a fluorescent fusion protein, is a dual function biosensor that has been utilized as a tool for the simultaneous measurement of intracellular chloride and pH in cells. ClopHensor has traditionally been used in conjunction with fluorescence microscopy for single cell measurements. Here, we present a promising multi-well format advancement for the use of ClopHensor as a potential high-throughput method capable of measuring fluorescence signal intensity across a well of confluent cells with highly reproducible results. Using this system, we gained mechanistic insight into an endogenous oxalate transporter in Chinese hamster ovary (CHO) cells expressing ClopHensor and the human chloride transporter, SLC26A3. SLC26A3, a known anion exchanger, has been proposed to play a role in colonic oxalate absorption in humans. Our attempt to study the role of SLC26A3 in oxalate transport revealed the presence of an endogenous oxalate transporter in CHO cells. This transporter was strongly inhibited by niflumate, and exhibited clear saturability. Use of ClopHensor in a multi-well cell assay allowed us to quickly demonstrate that the endogenous oxalate transporter was unable to exchange chloride for bicarbonate, unlike SLC26A3.

Pathophysiology and Treatment of Hyperoxaluria

Humans cannot degrade oxalate. Thus, oxalate that is generated in the liver and/or absorbed from the intestine must be eliminated by the kidneys. Among genetic causes, primary hyperoxaluria (PH) type 1 is the most common and occurs due to deficiency of hepatic peroxisomal alanine glyoxalate aminotransferase. PH2 is caused by deficiency of lysosomal glyoxalate reductase or hydroxypyruvate reductase, whereas PH3 results from deficiency of mitochondrial 4-hydroxy-2-oxoglutarate aldolase. Enteric hyperoxaluria is caused by excessive colonic oxalate absorption due to any type of fat malabsorption. The diagnosis of hyperoxaluria is based on the history, 24-hour urine studies, and genetic testing. Early diagnosis and timely intervention are essential. To treat PH, adequate fluid intake, inhibitors of calcium oxalate crystallization (citrate or neutral phosphorus), and pyridoxine-in responsive patients are all important. Intensive dialysis and prompt kidney or combined kidney-liver transplantation are essential to minimize systemic oxalosis if renal failure occurs. Dietary modifications (low fat, low oxalate, and adequate calcium) are key for enteric hyperoxaluria. Calcium can be used as an oxalate binder. Newer modalities including oxalate degrading bacteria, oral oxalate decarboxylase preparations, and inhibitory ribonucleic acids are all under investigation. This review contains 9 figures, 6 tables, and 90 references. Key Words: bariatric surgery, calcium oxalate, dialysis, enteric hyperoxaluria, fat malabsorption, genetic testing, kidney stone, nephrolithiasis, oxalate, oxalate decarboxylase, Oxalobacter formigenes, primary hyperoxaluria, pyridoxine, transplantation, urolithiasis

Robustness of the non-neuronal cholinergic system in rat large intestine against luminal challenges.

Acetylcholine and atypical esters of choline such as propionyl- and butyrylcholine are produced by the colonic epithelium and are released when epithelial receptors for short-chain fatty acids (SCFA) are stimulated by propionate. It is assumed that the SCFA used by the choline acetyltransferase (ChAT), the central enzyme for the production of these choline esters, originate from the colonic lumen, where they are synthesized during the bacterial fermentation of carbohydrates. Therefore, it seemed to be of interest to study whether the non-neuronal cholinergic system in the colonic epithelium is affected by maneuvers intended to stimulate or to inhibit colonic fermentation by changing the intestinal microbiota. In two series of experiments, rats were either fed with a high fiber diet (15.5% (w/v) crude fibers in comparison to 4.6% (w/w) in the control diet) or treated orally with the antibiotic vancomycin. High fiber diet induced an unexpected decrease in the luminal concentration of SCFA in the colon, but an increase in the caecum, suggesting an upregulation of colonic SCFA absorption, whereas vancomycin treatment resulted in the expected strong reduction of SCFA concentration in colon and caecum. MALDI MS analysis revealed a decrease in the colonic content of propionylcholine by high fiber diet and by vancomycin. High fiber diet caused a significant downregulation of ChAT expression on protein and mRNA level. Despite a modest increase in tissue conductance during the high fiber diet, main barrier and transport properties of the epithelium such as basal short-circuit current (Isc), the flux of the paracellularly transported marker, fluorescein, or the Isc induced by epithelial acetylcholine release evoked by propionate remained unaltered. These results suggest a remarkable stability of the non-neuronal cholinergic system in colonic epithelium against changes in the luminal environment underlying its biological importance for intestinal homeostasis.