pH-Dependent Protein Binding Properties of Uremic Toxins In Vitro.

Protein-bound uremic toxins (PBUTs) are difficult to remove using conventional dialysis treatment owing to their high protein-binding affinity. As pH changes the conformation of proteins, it may be associated with the binding of uremic toxins. Albumin conformation at pH 2 to 13 was analyzed using circular dichroism. The protein binding behavior between indoxyl sulfate (IS) and albumin was examined using isothermal titration calorimetry. Albumin with IS, and serum with IS, p-cresyl sulfate, indole acetic acid or phenyl sulfate, as well as serum from hemodialysis patients, were adjusted pH of 3 to 11, and the concentration of the free PBUTs was measured using mass spectrometry. Albumin was unfolded at pH < 4 or >12, and weakened interaction with IS occurred at pH < 5 or >10. The concentration of free IS in the albumin solution was increased at pH 4.0 and pH 11.0. Addition of human serum to each toxin resulted in increased free forms at acidic and alkaline pH. The pH values of serums from patients undergoing hemodialysis adjusted to 3.4 and 11.3 resulted in increased concentrations of the free forms of PBUTs. In conclusion, acidic and alkaline pH conditions changed the albumin conformation and weakened the protein binding property of PBUTs in vitro.

Can curcumin supplementation reduce plasma levels of gut-derived uremic toxins in hemodialysis patients? A pilot randomized, double-blind, controlled study.

BACKGROUND: Gut dysbiosis is common in patients with chronic kidney disease (CKD) and is closely related to inflammatory processes. Some nutritional strategies, such as bioactive compounds present in curcumin, have been proposed as an option to modulate the gut microbiota and decrease the production of uremic toxins such as indoxyl sulfate (IS), p-cresyl sulfate (pCS) and indole-3 acetic acid (IAA). OBJECTIVE: To evaluate the effects of curcumin supplementation on uremic toxins plasma levels produced by gut microbiota in patients with CKD on hemodialysis (HD). METHODS: Randomized, double-blind trial in 28 patients [53.6 ± 13.4 years, fourteen men, BMI 26.7 ± 3.7 kg/m2, dialysis vintage 37.5 (12-193) months]. Fourteen patients were randomly allocated to the curcumin group and received 100 mL of orange juice with 12 g carrot and 2.5 g of turmeric and 14 patients to the control group who received the same juice but without turmeric three times per week after HD sessions for three months. IS, pCS, IAA plasma levels were measured by reverse-phase high-performance liquid chromatography RESULTS: After three months of supplementation, the curcumin group showed a significant decrease in pCS plasma levels [from 32.4 (22.1-45.9) to 25.2 (17.9-37.9) mg/L, p = 0.009], which did not occur in the control group. No statistical difference was observed in IS and IAA levels in both groups. CONCLUSION: The oral supplementation of curcumin for three months seems to reduce p-CS plasma levels in HD patients, suggesting a gut microbiota modulation.

Factors of microinflammation in non-diabetic chronic kidney disease: a pilot study.

BACKGROUND: The relationships between digestive bacterial translocation, uremic toxins, oxidative stress and microinflammation in a population of chronic kidney disease (CKD) patients without metabolic nor inflammatory disease are unknown. METHODS: Bacterial translocation, uremic toxins, oxidative stress, and inflammation were assessed by measuring plasma levels of 16S ribosomal DNA (16S rDNA), p-cresyl sulfate (PCS), indoxyl sulfate (IS), indole acetic acid (IAA), F2-isoprostanes, hsCRP and receptor I of TNFα (RITNFα) in patients without metabolic nor inflammatory disease. 44 patients with CKD from stage IIIB to V and 14 controls with normal kidney function were included from the nephrology outpatients. 11 patients under hemodialysis (HD) were also included. Correlations between each factor and microinflammation markers were studied. RESULTS: 16S rDNA levels were not increased in CKD patients compared to controls but were decreased in HD compared to non-HD stage V patients (4.7 (3.9-5.3) vs 8.6 (5.9-9.7) copies/µl, p = 0.002). IS, PCS and IAA levels increased in HD compared to controls (106.3 (73.3-130.4) vs 3.17 (2.4-5.1) µmol/l, p < 0.0001 for IS; 174.2 (125-227.5) vs 23.7 (13.9-52.6) µmol/l, p = 0.006 for PCS; and 3.7 (2.6-4.6) vs 1.3 (1.0-1.9) µmol/l, p = 0.0002 for IAA). Urea increased in non-HD stage V patients compared to controls (27.6 (22.7-30.9) vs 5.4 (4.8-6.4) mmol/l, p < 0.0001) and was similar in HD and in non-HD stage V (19.3 (14.0-24.0) vs 27.6 (22.7-30.9) mmol/l, p = 0.7). RITNFα levels increased in HD patients compared to controls (12.6 (9.6-13.3) vs 1.1 (1.0-1.4) ng/ml, p < 0.0001); hsCRP levels increased in non-HD stage V patients compared to controls (2.9 (1.4-8.5) vs 0.8 (0.5-1.7) mg/l, p = 0.01) and remained stable in HD patients (2.9 (1.4-8.5) vs 5.1 (0.9-11.5) mg/l, p = 1). F2-isoprostanes did not differ in CKD patients compared to controls. Among uremic toxins, IS and urea were correlated to RITNFα (r = 0.8, p < 0.0001 for both). PCS, IS and urea were higher in patients with hsCRP≧5 mg/l (p = 0.01, 0.04 and 0.001 respectively). 16S rDNA, F2-isoprostanes were not correlated to microinflammation markers in our study. CONCLUSIONS: In CKD patients without any associated metabolic nor inflammatory disease, only PCS, IS, and urea were correlated with microinflammation. Bacterial translocation was decreased in patients under HD and was not correlated to microinflammation.

Serum Levels and Removal by Haemodialysis and Haemodiafiltration of Tryptophan-Derived Uremic Toxins in ESKD Patients.

Tryptophan is an essential dietary amino acid that originates uremic toxins that contribute to end-stage kidney disease (ESKD) patient outcomes. We evaluated serum levels and removal during haemodialysis and haemodiafiltration of tryptophan and tryptophan-derived uremic toxins, indoxyl sulfate (IS) and indole acetic acid (IAA), in ESKD patients in different dialysis treatment settings. This prospective multicentre study in four European dialysis centres enrolled 78 patients with ESKD. Blood and spent dialysate samples obtained during dialysis were analysed with high-performance liquid chromatography to assess uremic solutes, their reduction ratio (RR) and total removed solute (TRS). Mean free serum tryptophan and IS concentrations increased, and concentration of IAA decreased over pre-dialysis levels (67%, 49%, -0.8%, respectively) during the first hour of dialysis. While mean serum total urea, IS and IAA concentrations decreased during dialysis (-72%, -39%, -43%, respectively), serum tryptophan levels increased, resulting in negative RR (-8%) towards the end of the dialysis session (p < 0.001), despite remarkable Trp losses in dialysate. RR and TRS values based on serum (total, free) and dialysate solute concentrations were lower for conventional low-flux dialysis (p < 0.001). High-efficiency haemodiafiltration resulted in 80% higher Trp losses than conventional low-flux dialysis, despite similar neutral Trp RR values. In conclusion, serum Trp concentrations and RR behave differently from uremic solutes IS, IAA and urea and Trp RR did not reflect dialysis Trp losses. Conventional low-flux dialysis may not adequately clear Trp-related uremic toxins while high efficiency haemodiafiltration increased Trp losses.

Untargeted Metabonomics of Genetically Modified Cows Expressing Lactoferrin Based on Serum and Milk.

Metabolites of serum and milk from genetically modified (GM) cows and contrast check (CK) cows were comparatively investigated. Serum and milk were collected from genetically modified (GM) cows and contrast check (CK) cows, and then, they were analyzed using ultraperformance liquid chromatography-mass spectrometry (UPLC-MS) and gas chromatography-mass spectrometry (GC-MS). Although the level of some blood biochemical indexes for GM cows was shifted up or down, they were generally in normal physiological condition. Serum samples from lactoferrin GM cows exhibited reduced levels of amino acids and elevated levels of indoleacetate, α-keto acids, long-chain fatty acids, etc. GM milk possessed elevated levels of pentose and amino sugar metabolites, including arabitol, xylulose, glucuronate, and N-acetylgalactosamine. Interestingly, some essential nutrients, such as certain unsaturated fatty acids (e.g., eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and docosapentaenoic acid (DPA)), and some necessary rare sugars were significantly upregulated. Compared to the CK group, a Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was conducted based on the increased or decreased metabolites identified in the serum and milk samples of the GM group. The results showed that the GM cows were in healthy condition and their milk has improved benefits for customers. The milk from genetically modified cows was found to be a promising milk source for producing recombinant human lactoferrin (rhLF) for human beings.

Effects of theabrownin on serum metabolites and gut microbiome in rats with a high-sugar diet.

Evidence has proven that the gut microbiota is an important environmental factor contributing to obesity by altering host energy harvest and storage. We performed a high-throughput 16S rDNA sequencing association study and serum metabolomics profiling in rats with a high-sugar diet. Our studies revealed that the high sugar diet reduced the diversity of cecal microorganisms, while the combination of theabrownin and the high sugar diet increased the diversity of cecal microorganisms and promoted reproduction of Alloprevotella, Coprostanoligenes_group, Bacteroides, Prevotellaceae_NK3B31_group, Desulfovibrio, Intestinimonas, Alistipes, Bifidobacterium, Phascolarctobacterium, Ruminococcaceae_UCG-010 and Staphylococcus. The combination also inhibited the growth of Lactobacillus, Prevotellaceae_Ga6A1_group and Tyzzerella. The Firmicutes/Bacteroidetes (F/B) ratio can be significantly reduced after the intervention of theabrownin in high sugar diet rats, and the reproduction of Bacteroides acidifaciens (BA) and Staphylococcus saprophyticus subsp. saprophyticus can be promoted. We found that the obesity-associated gut microbial species were linked to the changes in circulating metabolites. Serum levels of deoxycholic acid, cholic acid, 1H-indole-3-acetic acid, 3-indole acrylic acid and melatonin were negatively correlated with BA and Staphylococcus saprophyticus subsp. saprophyticus, but positively correlated with Lactobacillus murinus, Leptum and Gut_metagenome. 2-Hydroxy-6-methylpyridin-3-carboxylic acid, l-homoserine, and 1,7-dimethylxanthine were positively correlated with BA and Staphylococcus saprophyticus subsp. saprophyticus, but negatively correlated with Lactobacillus murinus, Leptum, and Gut_metagenome. In a high sugar diet mode, theabrownin reduced the body weight and triglycerides and improved insulin resistance mainly by targeting the reproduction of intestinal microorganisms such as BA, Staphylococcus saprophyticus subsp. saprophyticus, Lactobacillus murinus, Leptum, Gut_metagenome and so on. A strong correlation between cecal microorganisms and serum metabolites, obesity and insulin resistance was observed. Theabrownin has high potential in reducing the risk of cardiovascular diseases such as diabetes and obesity.

Selective Transport of Protein-Bound Uremic Toxins in Erythrocytes.

To better understand the kinetics of protein-bound uremic toxins (PBUTs) during hemodialysis (HD), we investigated the distribution of hippuric acid (HA), indole-3-acetic acid (IAA), indoxyl sulfate (IS), and p-cresyl sulfate (pCS) in erythrocytes of HD patients. Their transport across the erythrocyte membrane was explored in the absence of plasma proteins in vitro in a series of loading and unloading experiments of erythrocytes from healthy subjects and HD patients, respectively. Furthermore, the impact of three inhibitors of active transport proteins in erythrocytes was studied. The four PBUTs accumulated in erythrocytes from HD patients. From loading and unloading experiments, it was found that (i) the rate of transport was dependent on the studied PBUT and increased in the following sequence: HA < IS < pCS < IAA and (ii) the solute partition of intra- to extra-cellular concentrations was uneven at equilibrium. Finally, inhibiting especially Band 3 proteins affected the transport of HA (both in loading and unloading), and of IS and pCS (loading). By exploring erythrocyte transmembrane transport of PBUTs, their kinetics can be better understood, and new strategies to improve their dialytic removal can be developed.

The Effect of Sevelamer on Serum Levels of Gut-Derived Uremic Toxins: Results from In Vitro Experiments and A Multicenter, Double-Blind, Placebo-Controlled, Randomized Clinical Trial.

High serum levels of gut-derived uremic toxins, especially p-cresyl sulfate (pCS), indoxyl sulfate (IS) and indole acetic acid (IAA), have been linked to adverse outcomes in patients with chronic kidney disease (CKD). Sevelamer carbonate could represent an interesting option to limit the elevation of gut-derived uremic toxins. The aim of the present study was to evaluate the adsorptive effect of sevelamer carbonate on different gut-derived protein-bound uremic toxins or their precursors in vitro, and its impact on the serum levels of pCS, IS and IAA in patients with CKD stage 3b/4. For the in vitro experiments, IAA, p-cresol (precursor of pCS) and indole (precursor of IS), each at a final concentration of 1 or 10 µg/mL, were incubated in centrifugal 30 kDa filter devices with 3 or 15 mg/mL sevelamer carbonate in phosphate-buffered saline at a pH adjusted to 6 or 8. Then, samples were centrifuged and free uremic toxins in the filtrates were analyzed. As a control experiment, the adsorption of phosphate was also evaluated. Additionally, patients with stage 3b/4 CKD (defined as an eGFR between 15 and 45 mL/min per 1.73 m2) were included in a multicenter, double-blind, placebo-controlled, randomized clinical trial. The participants received either placebo or sevelamer carbonate (4.8 g) three times a day for 12 weeks. The concentrations of the toxins and their precursors were measured using a validated high-performance liquid chromatography method with a diode array detector. In vitro, regardless of the pH and concentration tested, sevelamer carbonate did not show adsorption of indole and p-cresol. Conversely, with 10 µg/mL IAA, use of a high concentration of sevelamer carbonate (15 mg/mL) resulted in a significant toxin adsorption both at pH 8 (mean reduction: 26.3 ± 3.4%) and pH 6 (mean reduction: 38.7 ± 1.7%). In patients with CKD stage 3b/4, a 12-week course of treatment with sevelamer carbonate was not associated with significant decreases in serum pCS, IS and IAA levels (median difference to baseline levels: -0.12, 0.26 and -0.06 µg/mL in the sevelamer group vs. 1.97, 0.38 and 0.05 µg/mL in the placebo group, respectively). Finally, in vitro, sevelamer carbonate was capable of chelating a gut-derived uremic toxin IAA but not p-cresol and indole, the precursors of pCS and IS in the gut. In a well-designed clinical study of patients with stage 3b/4 CKD, a 12-week course of treatment with sevelamer carbonate was not associated with significant changes in the serum concentrations of pCS, IS and IAA.

Indole-3 acetic acid increased risk of impaired cognitive function in patients receiving hemodialysis.

Patients receiving hemodialysis (HD) have a higher risk of cognitive impairment and dementia than the general population. The accumulation of uremic toxins in the brain causes uremic encephalopathy, however, limited data exists to elucidate the effect of protein-bound uremic toxins on cognitive function. Here we investigate the effect of indole-3 acetic acid (IAA) and hippuric acid (HA), two different protein-bound uremic toxins from amino acid derivatives, on cognitive function by Silico and in a clinical study. Prevalent HD patients were enrolled in two independent hospitals. Serum IAA and HA were measured using mass spectrometry. Cognitive performance was measured using Mini-Mental State Examination (MMSE), Montreal Cognitive Assessment (MoCA), and Cognitive Abilities Screening Instrument (CASI) by trained psychologists. Using silico data to predict the effect of blood-brain barrier penetration was performed. The silico data demonstrated that IAA and HA had positive blood-brain barrier penetration ability. Amongst the 230 HD patients, serum IAA was associated with poor MMSE score (ß= -0.90, 95% CI -1.61 to -0.19) and poor CASI score (ß= -3.29, 95% CI -5.69 to -0.88) in stepwise multiple linear regression analysis. In logistic regression model, Serum IAA was also associated with cognitive impairment based on MMSE definition (OR, 1.96, 95% CI 1.10, 3.5) and CASI definition (OR, 2.09, 95% CI 1.21, 3.61). There was no correlation between Serum HA levels and cognitive function status. In conclusion, IAA, not HA, was associated with cognitive impairment in HD patients. Further large scale and prospective studies are needed to confirm our findings.

Association between Protein-Bound Uremic Toxins and Asymptomatic Cardiac Dysfunction in Patients with Chronic Kidney Disease.

Although the relationship between protein-bound uremic toxins (PBUTs) and cardiac structure and cardiac mortality in chronic kidney disease (CKD) has been studied in the past, the association between cardiac dysfunction and PBUTs has not yet been studied. We therefore evaluated the association between impaired peak cardiac performance and the serum free and total concentrations of potentially cardiotoxic PBUTs. In a cross-sectional study of 56 male CKD patients (stages 2⁻5 (pre-dialysis)) who were asymptomatic with no known cardiac diseases or diabetes we measured peak cardiac power (CPOmax), aerobic exercise capacity (VO2max), and echocardiographic parameters of cardiac morphology and evaluated their association with PBUTs. The serum total and free concentrations of indoxyl sulfate (IXS), p-cresyl sulfate (PCS), p-cresyl glucuronide, indole acetic acid, and hippuric acid showed significant negative correlation with CPOmax and VO2max. IXS and PCS were independently associated with CPOmax and VO2max even after controlling for eGFR. No correlation between left ventricular mass index (LVMI) and PBUTs was seen. The present study for the first time has demonstrated the association between subclinical cardiac dysfunction in CKD and serum levels of a panel of PBUTs. Further studies are required to evaluate the mechanism of cardiotoxicity of the individual uremic toxins.

The Impact of Uremic Toxicity Induced Inflammatory Response on the Cardiovascular Burden in Chronic Kidney Disease.

Uremic toxin (UT) retention in chronic kidney disease (CKD) affects biological systems. We aimed to identify the associations between UT, inflammatory biomarkers and biomarkers of the uremic cardiovascular response (BUCVR) and their impact on cardiovascular status as well as their roles as predictors of outcome in CKD patients. CKD patients stages 3, 4 and 5 (n = 67) were recruited and UT (indoxyl sulfate/IS, p-cresil sulfate/pCS and indole-3-acetic acid/IAA); inflammatory biomarkers [Interleukin-6 (IL-6), high sensitivity C reactive protein (hsCRP), monocyte chemoattractant protein-1 (MCP-1), soluble vascular adhesion molecule-1 (sVCAM-1), soluble intercellular adhesion molecule-1 (sICAM-1) and soluble Fas (sFas)] and BUCVRs [soluble CD36 (sCD36), soluble receptor for advanced glycation end products (sRAGE), fractalkine] was measured. Patients were followed for 5.2 years and all causes of death was used as the primary outcome. Artery segments collected at the moment of transplantation were used for the immunohistochemistry analysis in a separate cohort. Estimated glomerular filtration rate (eGFR), circulating UT, plasma biomarkers of systemic and vascular inflammation and BUCVR were strongly interrelated. Patients with plaque presented higher signs of UT-induced inflammation and arteries from CKD patients presented higher fractalkine receptor (CX3CR1) tissue expression. Circulating IS (p = 0.03), pCS (p = 0.007), IL-6 (p = 0.026), sFas (p = 0.001), sCD36 (p = 0.01) and fractalkine (p = 0.02) were independent predictors of total mortality risk in CKD patients. Our results reinforce the important role of uremic toxicity in the pathogenesis of cardiovascular disease (CVD) in CKD patients through an inflammatory pathway.

Does Low-Protein Diet Influence the Uremic Toxin Serum Levels From the Gut Microbiota in Nondialysis Chronic Kidney Disease Patients?

OBJECTIVES: To evaluate the effects of low-protein diet (LPD) on uremic toxins and the gut microbiota profile in nondialysis chronic kidney disease (CKD) patients. DESIGN AND METHODS: Longitudinal study with 30 nondialysis CKD patients (stage 3-4) undergoing LPD for 6 months. Adherence to the diet was evaluated based on the calculation of protein equivalent of nitrogen appearance from the 24-hour urine analysis. Good adherence to LPD was considered when protein intake was from 90% to 110% of the prescribed amount (0.6 g/kg/day). Food intake was analyzed by the 24-hour recall method. The anthropometric, biochemical and lipid profile parameters were measured according to standard methods. Uremic toxin serum levels (indoxyl sulfate, p-cresyl sulfate, indole-3-acetic acid) were obtained by reversed-phase high-performance liquid chromatography (RP-HPLC). Fecal samples were collected to evaluate the gut microbiota profile through polymerase chain reaction and denaturing gradient gel electrophoresis. Statistical analysis was performed by the SPSS 23.0 program software. RESULTS: Patients who adhered to the diet (n = 14) (0.7 ± 0.2 g/kg/day) presented an improvement in renal function (nonsignificant) and reduction in total and low-density lipoprotein cholesterol (183.9 ± 48.5-155.7 ± 37.2 mg/dL, P = .01; 99.4 ± 41.3-76.4 ± 33.2 mg/dL, P = .01, respectively). After 6 months of nutricional intervention, p-cresyl sulfate serum levels were reduced significantly in patients who adhered to the LPD (19.3 [9.6-24.7] to 15.5 [9.8-24.1] mg/L, P = .03), and in contrast, the levels were increased in patients who did not adhere (13.9 [8.0-24.8] to 24.3 [8.1-39.2] mg/L, P = .004). In addition, using the denaturing gradient gel electrophoresis technique, it was observed change in the intestinal microbiota profile after LPD intervention in both groups, and the number of bands was positively associated with protein intake (r = 0.44, P = .04). CONCLUSION: LPD seems be a good strategy to reduce the uremic toxins production by the gut microbiota in nondialysis CKD patients.

Probiotic Supplementation in Chronic Kidney Disease: A Double-blind, Randomized, Placebo-controlled Trial.

OBJECTIVE: The objective of the study was to evaluate the effects of probiotic supplementation on the gut microbiota profile and inflammatory markers in chronic kidney disease patients undergoing maintenance hemodialysis (HD). DESIGN AND METHODS: This was a randomized, double-blind, placebo-controlled study. Forty-six HD patients were assigned to receive 1 of 2 treatments: probiotic (n = 23; Streptococcus thermophilus, Lactobacillus acidophilus e Bifidobacterialongum, 90 billion colony-forming units per day) or placebo (n = 23) daily for 3 months. Blood and feces were collected at baseline and after intervention. The inflammatory markers (C-reactive protein and interleukin-6) were analyzed by immunoenzymatic assay (enzyme-linked immunosorbent assay). Uremic toxins plasma levels (indoxyl sulfate, p-cresyl sulfate, and indole-3-acetic acid) were obtained by Reversed-Phase High-Performance Liquid Chromatography. Routine laboratory parameters were measured by standard techniques. Fecal pH was measured by the colorimetric method, and the gut microbiota profile was assessed by Denaturing Gradient Gel Electrophoresis analysis. RESULTS: Sixteen patients remained in the probiotic group (11 men, 53.6 ± 11.0 year old, 25.3 ± 4.6 kg/m2) and 17 in the placebo group (10 men, 50.3 ± 8.5 year old, 25.2 ± 5.7 kg/m2). After probiotic supplementation there was a significant increase in serum urea (from 149.6 ± 34.2 mg/dL to 172.6 ± 45.0 mg/dL, P = .02), potassium (from 4.4 ± 0.4 mmol/L to 4.8 ± 0.4 mmol/L, P = .02), and indoxyl sulfate (from 31.2 ± 15.9 to 36.5 ± 15.0 mg/dL, P = .02). The fecal pH was reduced from 7.2 ± 0.8 to 6.5 ± 0.5 (P = .01). These parameters did not change significantly in placebo group. Changes in the percentage delta (Δ) between groups were exhibited with no statistical differences observed. The inflammatory markers and gut profile were not altered by supplementation. CONCLUSIONS: Aprobiotic supplementation failed to reduce uremic toxins and inflammatory markers. Therefore, probiotic therapy should be chosen with caution in HD patients. Further studies addressing probiotic therapy in chronic kidney disease patients are needed.

Adsorption of Protein-Bound Uremic Toxins Through Direct Hemoperfusion With Hexadecyl-Immobilized Cellulose Beads in Patients Undergoing Hemodialysis.

An accumulation of protein-bound uremic toxins (PBUTs) is one of major reasons for development of uremia-related complications. We examined the PBUT removal ability of a hexadecyl-immobilized cellulose bead (HICB)-containing column for patients undergoing hemodialysis. Adsorption of indoxyl sulfate (IS), a representative PBUT, to HICBs was examined in vitro. The HICB column was used in patients undergoing hemodialysis for direct hemoperfusion with a regular hemodialyzer. The serum IS, indole acetic acid (IAA), phenyl sulfate (PhS), and p-cresyl sulfate (PCS) levels were measured before and after passing the column. HICBs adsorbed protein-free (free) IS in a dose- and time-dependent manner in vitro (55.4 ± 1.4% adsorption of 1 millimolar, 251 µg/mL, IS for 1 h). In clinical studies, passing the HICB-containing column decreased the serum level of free IS, IAA, PhS, and PCS levels significantly (by 34.4 ± 30.0%, 34.8 ± 25.4%, 28.4 ± 18.0%, and 34.9 ± 22.1%, respectively), but not protein-bound toxins in maintenance hemodialysis patients. HICBs absorbed some amount of free PBUTs, but the clinical trial to use HICB column did not show effect to reduce serum PBUTs level in hemodialysis patients. Adsorption treatment by means of direct hemoperfusion with regular hemodialysis may become an attractive blood purification treatment to increase PBUT removal when more effective materials to adsorb PBUTs selectively will be developed.

Indole 3-acetic acid, indoxyl sulfate and paracresyl-sulfate do not influence anemia parameters in hemodialysis patients.

BACKGROUND: The main reason for anemia in renal failure patients is the insufficient erythropoietin production by the kidneys. Beside erythropoietin deficiency, in vitro studies have incriminated uremic toxins in the pathophysiology of anemia but clinical data are sparse. In order to assess if indole 3-acetic acid (IAA), indoxyl sulfate (IS), and paracresyl sulfate (PCS) -three protein bound uremic toxins- are clinically implicated in end-stage renal disease anemia we studied the correlation between IAA, IS and PCS plasmatic concentrations with hemoglobin and Erythropoietin Stimulating Agents (ESA) use in hemodialysis patients. METHODS: Between June and July 2014, we conducted an observational cross sectional study in two hemodialysis center. Three statistical approaches were conducted. First, we compared patients treated with ESA and those not treated. Second, we performed linear regression models between IAA, IS, and PCS plasma concentrations and hemoglobin, the ESA dose over hemoglobin ratio (ESA/Hemoglobin) or the ESA resistance index (ERI). Third, we used a polytomous logistic regression model to compare groups of patients with no/low/high ESA dose and low/high hemoglobin statuses. RESULTS: Overall, 240 patients were included in the study. Mean age ± SD was 67.6 ± 16.0 years, 55.4% were men and 42.5% had diabetes mellitus. When compared with ESA treated patients, patients with no ESA had higher hemoglobin (mean 11.4 ± 1.1 versus 10.6 ± 1.2 g/dL; p <0.001), higher transferrin saturation (TSAT, 31.1 ± 16.3% versus 23.1 ± 11.5%; p < 0.001), less frequently an IV iron prescription (52.1 versus 65.7%, p = 0.04) and were more frequently treated with hemodiafiltration (53.5 versus 36.7%). In univariate analysis, IAA, IS or PCS plasma concentrations did not differ between the two groups. In the linear model, IAA plasma concentration was not associated with hemoglobin, but was negatively associated with ESA/Hb (p = 0.02; R = 0.18) and with the ERI (p = 0.03; R = 0.17). IS was associated with none of the three anemia parameters. PCS was positively associated with hemoglobin (p = 0.03; R = 0.14), but negatively with ESA/Hb (p = 0.03; R = 0.17) and the ERI (p = 0.02; R = 0.19). In multivariate analysis, the association of IAA concentration with ESA/Hb or ERI was not statistically significant, neither was the association of PCS with ESA/Hb or ERI. Identically, in the subgroup of 76 patients with no inflammation (CRP <5 mg/L) and no iron deficiency (TSAT >20%) linear regression between IAA, IS or PCS and any anemia parameter did not reach significance. In the third model, univariate analysis showed no intergroup significant differences for IAA and IS. Regarding PCS, the Low Hb/High ESA group had lower concentrations. However, when we compared PCS with the other significant characteristics of the five groups to the Low Hb/high ESA (our reference group), the polytomous logistic regression model didn't show any significant difference for PCS. CONCLUSIONS: In our study, using three different statistical models, we were unable to show any correlation between IAA, IS and PCS plasmatic concentrations and any anemia parameter in hemodialysis patients. Indolic uremic toxins and PCS have no or a very low effect on anemia parameters.

Magnolol, a Natural Polyphenol, Attenuates Dextran Sulfate Sodium-Induced Colitis in Mice.

Magnolol is a lignan with anti-inflammatory activity identified in Magnolia officinalis. Ulcerative colitis (UC), one of the types of inflammatory bowel disease (IBD), is a disease that causes inflammation and ulcers in the colon. To investigate the effect of magnolol in dextran sulfate sodium (DSS)-induced experimental UC model, male C57 mice were treated with 2% DSS drinking water for 5 consecutive days followed by intragastric administration with magnolol (5, 10 and 15 mg/kg) daily for 7 days. The results showed that magnolol significantly attenuated disease activity index, inhibited colonic shortening, reduced colonic lesions and suppressed myeloperoxidase (MPO) activity. Moreover, colonic pro-inflammatory cytokines (TNF-α, IL-6, and IL-1ß) induced by colitis were dramatically decreased by magnolol. To further unveil the metabolic signatures upon magnolol treatment, mass spectrometry-based metabolomic analysis of the small molecular metabolites in mice serum were performed. Compared with controls, abnormality of serum metabolic phenotypes in DSS-treated mice were effectively reversed by different doses of magnolol. In particular, magnolol treatment effectively elevated the serum levels of tryptophan metabolites including kynurenic acid (KA), 5-hydroxyindoleacetic acid, indoleacetic acid (IAA), indolelactic acid and indoxylsulfuric acid, which are potential aryl hydrocarbon receptor (AHR) ligands to impact colitis. These findings suggest that magnolol exerts anti-inflammatory effect on DSS-induced colitis and its underlying mechanisms are associated with the restoring of tryptophan metabolites that inhibit the colonic inflammation.

Absorption, Distribution, Metabolism, and Excretion of the Oral Prostaglandin D2 Receptor 2 Antagonist Fevipiprant (QAW039) in Healthy Volunteers and In Vitro.

Fevipiprant is a novel oral prostaglandin D2 receptor 2 (DP2; also known as CRTh2) antagonist, which is currently in development for the treatment of severe asthma and atopic dermatitis. We investigated the absorption, distribution, metabolism, and excretion properties of fevipiprant in healthy subjects after a single 200-mg oral dose of [14C]-radiolabeled fevipiprant. Fevipiprant and metabolites were analyzed by liquid chromatography coupled to tandem mass spectrometry and radioactivity measurements, and mechanistic in vitro studies were performed to investigate clearance pathways and covalent plasma protein binding. Biotransformation of fevipiprant involved predominantly an inactive acyl glucuronide (AG) metabolite, which was detected in plasma and excreta, representing 28% of excreted drug-related material. The AG metabolite was found to covalently bind to human plasma proteins, likely albumin; however, in vitro covalent binding to liver protein was negligible. Excretion was predominantly as unchanged fevipiprant in urine and feces, indicating clearance by renal and possibly biliary excretion. Fevipiprant was found to be a substrate of transporters organic anion transporter 3 (OAT3; renal uptake), multidrug resistance gene 1 (MDR1; possible biliary excretion), and organic anion-transporting polypeptide 1B3 (OATP1B3; hepatic uptake). Elimination of fevipiprant occurs via glucuronidation by several uridine 5'-diphospho glucuronosyltransferase (UGT) enzymes as well as direct excretion. These parallel elimination pathways result in a low risk of major drug-drug interactions or pharmacogenetic/ethnic variability for this compound.

Gut microbiome-related metabolic changes in plasma of antibiotic-treated rats.

The intestinal microbiota contributes to the metabolism of its host. Adequate identification of the microbiota's impact on the host plasma metabolites is lacking. As antibiotics have a profound effect on the microbial composition and hence on the mammalian-microbiota co-metabolism, we studied the effects of antibiotics on the "functionality of the microbiome"-defined as the production of metabolites absorbed by the host. This metabolomics study presents insights into the mammalian-microbiome co-metabolism of endogenous metabolites. To identify plasma metabolites related to microbiome changes due to antibiotic treatment, we have applied broad-spectrum antibiotics belonging to the class of aminoglycosides (neomycin, gentamicin), fluoroquinolones (moxifloxacin, levofloxacin) and tetracyclines (doxycycline, tetracycline). These were administered orally for 28 days to male rats including blood sampling for metabolic profiling after 7, 14 and 28 days. Fluoroquinolones and tetracyclines can be absorbed from the gut; whereas, aminoglycosides are poorly absorbed. Hippuric acid, indole-3-acetic acid and glycerol were identified as key metabolites affected by antibiotic treatment, beside changes mainly concerning amino acids and carbohydrates. Inter alia, effects on indole-3-propionic acid were found to be unique for aminoglycosides, and on 3-indoxylsulfate for tetracyclines. For each class of antibiotics, specific metabolome patterns could be established in the MetaMap®Tox data base, which contains metabolome data for more than 550 reference compounds. The results suggest that plasma-based metabolic profiling (metabolomics) could be a suitable tool to investigate the effect of antibiotics on the functionality of the microbiome and to obtain insight into the mammalian-microbiome co-metabolism.

Tryptophan metabolism, its relation to inflammation and stress markers and association with psychological and cognitive functioning: Tasmanian Chronic Kidney Disease pilot study.

BACKGROUND: Adults with chronic kidney disease (CKD) exhibit alterations in tryptophan metabolism, mainly via the kynurenine pathway, due to higher enzymatic activity induced mainly by inflammation. Indoles produced by gut-microflora are another group of tryptophan metabolites related to inflammation and conditions accompanying CKD. Disruptions in tryptophan metabolism have been associated with various neurological and psychological disorders. A high proportion of CKD patients self-report symptoms of depression and/or anxiety and decline in cognitive functioning. This pilot study examines tryptophan metabolism in CKD and explores associations with psychological and cognitive functioning. METHODS: Twenty-seven adults with CKD were part of 49 patients recruited to participate in a prospective pilot study, initially with an eGFR of 15-29 mL/min/1.73 m2. Only participants with viable blood samples and complete psychological/cognitive data at a 2-year follow-up were included in the reported cross-sectional study. Serum samples were analysed by Liquid Chromatography coupled to Mass Spectrometry, for tryptophan, ten of its metabolites, the inflammation marker neopterin and the hypothalamic-pituitary-adrenal (HPA) axis marker cortisol. RESULTS: The tryptophan breakdown index (kynurenine / tryptophan) correlated with neopterin (Pearson R = 0.51 P = 0.006) but not with cortisol. Neopterin levels also correlated with indoxyl sulfate (R = 0.68, P < 0.0001) and 5 metabolites of tryptophan (R range 0.5-0.7, all P ≤ 0.01), which were all negatively related to eGFR (P < 0.05). Higher levels of kynurenic acid were associated with lower cognitive functioning (Spearman R = -0.39, P < 0.05), while indole-3 acetic acid (IAA) was correlated with anxiety and depression (R = 0.52 and P = 0.005, R = 0.39 and P < 0.05, respectively). CONCLUSIONS: The results of this preliminary study suggest the involvement of inflammation in tryptophan breakdown via the kynurenine pathway, yet without sparing tryptophan metabolism through the 5-HT (serotonin) pathway in CKD patients. The multiple moderate associations between indole-3 acetic acid and psychological measures were a novel finding. The presented pilot data necessitate further exploration of these associations within a large prospective cohort to assess the broader significance of these findings.

Correlations between Plasma Levels of Anionic Uremic Toxins and Clinical Parameters in Hemodialysis Patients.

When the kidney is seriously impaired, various uremic toxins (UTs) accumulate in the body, often exerting unfavorable effects on physiological functions and drug pharmacokinetics. To prevent this, it is important to determine plasma UT levels accurately in chronic kidney disease patients. Although attempts to predict plasma UT levels using biomarkers have been made, the correlation between UT levels and the markers is not yet fully understood. In this study, we assessed the correlations among plasma levels of indoxyl sulfate (IS), indoleacetic acid (IA), and 3-carboxy-4-methyl-5-propyl-2-furanpropionic acid (CMPF) in 20 hemodialysis patients and evaluated the relationship between the plasma levels of UTs and clinical parameters, such as serum creatinine (Scr), blood urea nitrogen, and estimated glomerular filtration rate (eGFR), with special focus on IS. There were no correlations among the plasma levels of the three UTs before and immediately after hemodialysis. However, a significant correlation was observed between plasma IS levels and Scr before hemodialysis (r=0.643, p=0.002), with the correlation becoming much stronger when using the data obtained immediately after hemodialysis (r=0.744, p<0.001). Further, plasma IS levels showed a significant negative correlation with eGFR (r=-0.558, p=0.011). However, no correlations were observed for IA or CMPF. The results obtained from this study suggest that plasma IS levels can be predicted from Scr values, although the precise mechanism behind the correlation remains to be clarified.