Fecal short chain fatty acids and urinary 3-indoxyl sulfate do not discriminate between patients with Crohn´s disease and ulcerative colitis and are not of diagnostic utility for predicting disease severity.

BACKGROUND: Urinary 3-indoxyl sulfate levels as well as fecal short chain fatty acid (SCFA) concentrations are surrogate markers for gut microbiota diversity. Patients with inflammatory bowel diseases (IBDs) and patients with primary sclerosing cholangitis (PSC), a disease closely associated with IBD, have decreased microbiome diversity. In this paper, the fecal SCFAs propionate, acetate, butyrate and isobutyrate of patients with IBD and patients with PSC-IBD and urinary 3-indoxyl sulfate of IBD patients were determined to study associations with disease etiology and severity. METHODS: SCFA levels in feces of 64 IBD patients and 20 PSC-IBD patients were quantified by liquid chromatography with tandem mass spectrometry (LC-MS/MS). Urinary 3-indoxyl sulfate levels of 45 of these IBD patients were analysed by means of reversed-phase liquid chromatography-electrospray ionization-tandem mass spectrometry. Feces of 17 healthy controls and urine of 13 of these controls were analyzed in parallel. These cohorts had comparable sex distribution and age. RESULTS: Urinary 3-indoxyl sulfate concentrations (normalized to urinary creatinine levels) was increased (P = 0.030) and fecal isobutyrate levels (normalized to dry weight of the stool sample) of IBD patients were decreased (P = 0.035) in comparison to healthy controls. None of the analyzed metabolites differed between patients with Crohn´s disease (CD) and patients with ulcerative colitis (UC). Fecal acetate and butyrate positively correlated with fecal calprotectin (P = 0.040 and P = 0.005, respectively) and serum C-reactive protein (P = 0.024 and P = 0.025, respectively) in UC but not CD patients. UC patients with fecal calprotectin levels above 150 µg/g, indicating intestinal inflammatory activity, had higher fecal acetate (P = 0.016), butyrate (P = 0.007) and propionate (P = 0.046) in comparison to patients with fecal calprotectin levels < 50 µg/g. Fecal SCFA levels of PSC-IBD and IBD patients were comparable. CONCLUSIONS: Current findings suggest that analysis of urinary 3-indoxyl-sulfate as well as fecal SCFAs has no diagnostic value for IBD and PSC-IBD diagnosis or monitoring of disease severity.

Specific Urinary Metabolites in Malignant Melanoma.

Background and objectives: Melanin, which has a confirmed role in melanoma cell behaviour, is formed in the process of melanogenesis and is synthesized from tryptophan, L-tyrosine and their metabolites. All these metabolites are easily detectable by chromatography in urine. Materials and Methods: Urine samples of 133 individuals (82 malignant melanoma patients and 51 healthy controls) were analysed by reversed-phase high-performance liquid chromatography (RP-HPLC). The diagnosis of malignant melanoma was confirmed histologically. Results: Chromatograms of melanoma patients showed increased levels of 5,6-dihydroxyindole-2-carboxylic acid, vanilmandelic acid, homovanilic acid, tryptophan, 5-hydroxyindole-3-acetic acid, and indoxyl sulphate compared to healthy controls. Concentration of indoxyl sulphate, homovanilic acid and tryptophan were significantly increased even in the low clinical stage 0 of the disease (indoxyl sulphate, homovanilic acid and tryptophan in patients with clinical stage 0 vs. controls expressed as medium/ interquartile range in µmol/mmol creatinine: 28.37/15.30 vs. 5.00/6.91; 47.97/33.08 vs. 7.33/21.25; and 16.38/15.98 vs. 3.46/6.22, respectively). Conclusions: HPLC detection of metabolites of L-tyrosine and tryptophan in the urine of melanoma patients may play a significant role in diagnostics as well as a therapeutic strategy of melanoma cancer.

Update of uremic toxin research by mass spectrometry.

Mass spectrometry (MS) has been successfully applied for the identification and quantification of uremic toxins and uremia-associated modified proteins. This review focuses on the recent progress in the MS analysis of uremic toxins. Uremic toxins include low-molecular weight solutes, protein-bound low-molecular weight solutes, and middle molecules (peptides and proteins). Based on MS analysis of these uremic toxins, the pathogenesis of the uremic symptoms will be elucidated to prevent and manage the symptoms. Notably, protein-bound uremic toxins such as indoxyl sulfate, p-cresyl sulfate, and 3-carboxy-4-methyl-5-propyl-2-furanpropionic acid have emerged as important targets of therapeutic removal. Hemodialysis even with a high-flux membrane cannot efficiently remove the protein-bound uremic toxins because of their high albumin-binding property. The accumulation of these protein-bound uremic toxins in the blood of dialysis patients might play an important role in the development of uremic complications such as cardiovascular disease. Indoxyl sulfate is the most promising protein-bound uremic toxin as a biomarker of progress in chronic kidney disease. Novel dialysis techniques or membranes should be developed to efficiently remove these protein-bound uremic toxins for the prevention and management of uremic complications.

Metabolomics analysis reveals elevation of 3-indoxyl sulfate in plasma and brain during chemically-induced acute kidney injury in mice: investigation of nicotinic acid receptor agonists.

An investigative renal toxicity study using metabolomics was conducted with a potent nicotinic acid receptor (NAR) agonist, SCH 900424. Liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS) techniques were used to identify small molecule biomarkers of acute kidney injury (AKI) that could aid in a better mechanistic understanding of SCH 900424-induced AKI in mice. The metabolomics study revealed 3-indoxyl sulfate (3IS) as a more sensitive marker of SCH 900424-induced renal toxicity than creatinine or urea. An LC-MS assay for quantitative determination of 3IS in mouse matrices was also developed. Following treatment with SCH 900424, 3IS levels were markedly increased in murine plasma and brain, thereby potentially contributing to renal- and central nervous system (CNS)-related rapid onset of toxicities. Furthermore, significant decrease in urinary excretion of 3IS in those animals due to compromised renal function may be associated with the elevation of 3IS in plasma and brain. These data suggest that 3IS has a potential to be a marker of renal and CNS toxicities during chemically-induced AKI in mice. In addition, based on the metabolomic analysis other statistically significant plasma markers including p-cresol-sulfate and tryptophan catabolites (kynurenate, kynurenine, 3-indole-lactate) might be of toxicological importance but have not been studied in detail. This comprehensive approach that includes untargeted metabolomic and targeted bioanalytical sample analyses could be used to investigate toxicity of other compounds that pose preclinical or clinical development challenges in a pharmaceutical discovery and development.

Reduction in protein-bound solutes unacceptable as marker of dialysis efficacy during alternate-night nocturnal hemodialysis.

BACKGROUND: The uremic retention solutes indoxyl sulfate and p-cresyl sulfate are linked to cardiovascular disease and overall survival. Dialytic clearances are limited, which is principally attributed to tight protein binding. Extending dialysis duration would be expected to substantially increase protein-bound uremic solute removal. The aim of the current study was to study protein-bound uremic retention solute clearances and kinetics during longer-hours nocturnal hemodialysis. METHODS: In a prospective cohort study of 32 maintenance alternate-night nocturnal hemodialysis patients, we followed serum concentrations, solute removals and solute clearances of p-cresyl sulfate and indoxyl sulfate. Spent dialysate sampling was fractionated to compare solute removals between the first 4 h and next 4 h of nocturnal dialysis. Single-compartment variable volume kinetics were calculated. RESULTS: Dialytic clearances of protein-bound uremic retention solutes are maintained during nocturnal (longer-hours) dialysis. Clearances of indoxyl sulfate exceed those of p-cresyl sulfate, presumably due to less tight protein-binding. Apparent distribution volumes increase substantially during nocturnal dialysis, indicative of multi-compartmental behavior of the protein-bound uremic retention solutes indoxyl sulfate and p-cresyl sulfate. CONCLUSIONS: During nocturnal hemodialysis, serum concentrations of protein-bound solute concentrations are reduced less than predicted. Reduction ratios are not a valid tool to estimate total solute removal of protein-bound uremic retention solutes.

Evaluation of Salivary Indoxyl Sulfate with Proteinuria for Predicting Graft Deterioration in Kidney Transplant Recipients.

Acute kidney injury (AKI) is a significant risk factor for developing chronic kidney disease and progression to end-stage renal disease in elderly patients. AKI is also a relatively common complication after kidney transplantation (KTx) associated with graft failure. Since the lifespan of a transplanted kidney is limited, the risk of the loss/deterioration of graft function (DoGF) should be estimated to apply the preventive treatment. The collection of saliva and urine is more convenient than collecting blood and can be performed at home. The study aimed to verify whether non-invasive biomarkers, determined in saliva and urine, may be useful in the prediction of DoGF in kidney transplant recipients (KTRs) (n = 92). Salivary and serum toxins (p-cresol sulfate, pCS; indoxyl sulfate, IS) concentrations were determined using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Urinary proteins, hemoglobin, and glucose were measured using a semi-quantitative strip test. Salivary IS (odds ratio (OR) = 1.19), and proteinuria (OR = 3.69) were demonstrated as independent factors for the prediction of DoGF. Satisfactory discriminatory power (area under the receiver operating characteristic curve (AUC) = 0.71 ± 0.07) and calibration of the model were obtained. The model showed that categories of the increasing probability of the risk of DoGF are associated with the decreased risk of graft survival. The non-invasive diagnostic biomarkers are a useful screening tool to identify high-risk patients for DoGF.

Indoxyl sulfate caused behavioral abnormality and neurodegeneration in mice with unilateral nephrectomy.

Chronic Kidney Disease (CKD) and neurodegenerative diseases are aging-related diseases. CKD with declined renal function is associated with an elevation of circulating indoxyl sulfate, a metabolite synthesized by gut microbes. We explored the roles of gut microbial metabolites in linking with Central Nervous System (CNS) diseases by administrating indoxyl sulfate intraperitoneally to male C57BL/6 mice with unilateral nephrectomy. Upon exposure, the accumulation of indoxyl sulfate was noted in the blood, prefrontal cortical tissues, and cerebrospinal fluid. Mice showed behavioral signs of mood disorders and neurodegeneration such as anxiety, depression, and cognitive impairment. Those behavioral changes were accompanied by disturbed neuronal survival, neural stem cell activity, expression of Brain-Derived Neurotrophic Factor, serotonin, corticosterone, and Repressor Element-1 Silencing Transcription Factor, and post-receptor intracellular signaling, as well as upregulated oxidative stress and neuroinflammation. Uremic toxin adsorbent AST-120 improved the above mentioned changes. Intriguingly, intracerebroventricular indoxyl sulfate administration only caused limited alterations in the normal mice and the alterations were reversed by aryl hydrocarbon receptor antagonism. The findings suggest pathogenic roles of indoxyl sulfate in the development of CNS diseases, and highlight gut microbiota as alternative targets for intervention with the aim of slowing down the progression of CKD and decreasing CNS complications.

HPLC study of uremic fluids related to optical dialysis adequacy monitoring.

PURPOSE: The aim of this study was to investigate uremia-related high-performance liquid chromatography (HPLC) ultraviolet (UV) absorbance profiles of serum and spent dialysate and to study the removal of uremic retention solutes in connection with optical dialysis adequacy monitoring. METHODS: 10 uremic patients were investigated using online spectrophotometry at a wavelength of 280 nm over the course of 30 hemodialysis treatments. The dialysate and blood samples were taken and analyzed simultaneously using standard biochemical methods and reversed-phase HPLC. Filters with cutoff at 3 kDa and 70 kDa were used for the pre-treatment of the serum. The chromatographic peaks were detected by a UV detector at wavelengths of 254 and 280 nm. RESULTS: This study indicated that the main solute responsible for UV absorbance in the spent dialysate is a low-molecular-weight, water-soluble, non-protein-bound compound uric acid (UA). Three additional uremic retention solutes - creatinine (CR), indoxyl sulphate (IS) and hippuric acid (HA) - were identified from the HPLC profiles. The number of detected HPLC peaks was not significantly different for a serum filtered through the 3 kDa or 70 kDa cutoff filters, and was lower for the spent dialysate, indicating that the molecular weight (MW) of the main UV chromophores in the uremic fluids did not exceed 3 kDa. The reduction ratio (RR) estimated by the total area of HPLC peaks at 254 nm and 280 nm in the serum and by the online UV absorbance at 280 nm was best related to the removal of small water-soluble non-protein bound solutes like urea (UR), CR and UA. CONCLUSIONS: The present study contributes new information on the removal of uremic retention solutes during hemodialysis and on the origin of the optical dialysis adequacy monitoring signal.

Removal of Urea, ß2-Microglobulin, and Indoxyl Sulfate Assessed by Absorbance and Fluorescence in the Spent Dialysate During Hemodialysis.

In this study, simultaneous removal assessment of marker molecules from three uremic toxin groups was performed during different hemodialysis treatment modalities using optical characteristics of spent dialysate. Results from optical measurements were compared with the results from chemical laboratory. Ten chronic dialysis patients, mean age 59 ± 15 years, were included in the study during 40 hemodialysis sessions. Low-flux hemodialysis (HD), high-flux hemodialysis (HF), and postdilutional online hemodiafiltration (HDF) with different settings were used. The reduction ratio (RR) and total removed solute (TRS) of three uremic solutes were determined: small molecular weight urea, middle molecular ß2-microglobulin (B2M), and protein-bound indoxyl sulfate (IS). Concentrations of these solutes in the spent dialysate were measured by laboratory (lab) and optical (opt) methods, in the serum by laboratory methods, and calculated RR values in percentage were compared accordingly. Total removed solute was obtained from the total dialysate collection (TDC) using lab and opt methods. The highest RR values were found for urea and B2M, and the lowest for IS. The difference between RR of lab and opt results estimated as mean accuracy (BIAS) was ≤8.1% for all three solutes. Good correspondence between TRS lab vs. opt was achieved, resulting in strong linear correlation values R from 0.727 for urea to 0.971 for IS. Accuracy for TRS values as BIAS ± standard error (SE), comparing lab vs. opt, showed no statistical difference for any of the observed uremic solutes (P > 0.05). The accuracy of the optical method was not influenced by the dialysis modality (HD, HF, and HDF).

Identification and characterisation of fluorescent substances in spent dialysis fluid.

Patients who suffer from end-stage renal disease require renal replacement therapy, including haemodialysis. While applying extracorporeal blood treatment, uraemic toxins accumulated in the patients' blood pass into a physiological solution, the dialysis fluid. Thus, important information about the patient's health status can be obtained by analysing the spent dialysis fluid. To make use of this information, corresponding analysis concepts must be developed. In this context, this article reports the analysis of fluorescence in spent dialysis fluid. Excitation and emission maxima of fluorescence in spent dialysis fluid were recorded, and the main fluorescent substances were identified and quantified using high-performance liquid chromatography analysis. Fluorescence in spent dialysis fluid has two prominent excitation maxima at λex1 = 228 nm and λex2 = 278 nm. However, both excitation maxima cause emission with maxima at λem = 350 nm. Identification of fluorescent substances using high-performance liquid chromatography showed that the main contributors to the overall fluorescence in spent dialysis fluid are tyrosine, tryptophan, indoxyl sulphate and indole-3-acetic acid. However, these substances are responsible for only one-third of the overall fluorescence of spent dialysis fluid. A large number of substances, each of which contributes only to a small part to the overall fluorescence, emit the remaining fluorescence.

Plasma indoxyl sulfate levels predict cardiovascular events in patients with mild chronic heart failure.

Indoxyl sulfate (IS) is associated with either chronic kidney disease or renal failure, which may predict cardiovascular events via cardiorenal syndrome. The present study aimed to elucidate whether the plasma levels of IS can predict the occurrence of cardiovascular events in patients with chronic heart failure (CHF) and investigate which causes of CHF leading to cardiovascular events are highly influenced by plasma IS levels. We measured the plasma IS levels in 165 patients with CHF [valvular disease: 78, dilated cardiomyopathy: 29, hypertrophic cardiomyopathy (HCM): 25 and others: 33] admitted to our hospital in 2012, and we followed up these patients for more than 5 years (the median follow-up period: 5.3 years). We measured the plasma IS level in 165 patients with CHF, and Kaplan-Meier analyses showed that high plasma IS levels (≥ 0.79 µg/mL, the median value) could predict the occurrence of cardiovascular events, i.e., cardiovascular death or rehospitalization due to the worsening of CHF. The sub-analyses showed that the high IS level could predict cardiovascular events in patients with CHF due to HCM and that the plasma IS levels were closely associated with left ventricular (LV) dimension, LV systolic dysfunction, and plasma B-type natriuretic peptide levels, rather than LV diastolic dysfunction. Plasma IS level predicts cardiovascular events in patients with CHF, especially those with HCM along with cardiac dysfunction. Besides, IS may become a proper biomarker to predict cardiovascular events in patients with CHF.

Relationship of impaired olfactory function in ESRD to malnutrition and retained uremic molecules.

BACKGROUND: Olfactory function is impaired in patients with end-stage renal disease (ESRD) and may contribute to uremic anorexia. Only limited correlations of olfactory function and nutritional status were reported. This study examines the relationship of impaired olfactory function to malnutrition and levels of the retained uremic solutes monomethylamine, ethylamine, indoxyl sulfate, and P-cresol sulfate. STUDY DESIGN: Cross-sectional observational study. SETTING & PARTICIPANTS: 31 stable maintenance hemodialysis patients from an urban outpatient dialysis unit and 18 people with normal renal function participated. PREDICTOR: Nutritional status assigned by using Subjective Global Assessment (SGA) score; SGA score of 7 indicates normal nutritional status; SGA score of 5 to 6, mild malnutrition; and SGA score of 3 to 4, moderate malnutrition. OUTCOMES & MEASUREMENTS: The primary outcome is olfactory function, assessed using the University of Pennsylvania Smell Identification Test. Levels of retained uremic solutes were measured from a predialysis serum sample. Demographic data and laboratory values for nutritional status, adequacy of dialysis, and inflammation were collected. RESULTS: Mean smell scores were 34.9 +/- 1.4 for controls, 33.5 +/- 3.3 for patients with SGA score of 7, 28.3 +/- 5.8 for patients with SGA score of 5 to 6, and 27.9 +/- 4.4 for patients with SGA score of 3 to 4 (P < 0.001 comparing healthy patients with all patients with ESRD). There was no difference in mean smell scores for healthy controls and patients with SGA score of 7. However, patients with lower smell scores had significantly lower SGA scores (P = 0.02) and higher C-reactive protein levels (P = 0.02). Neither smell score nor nutritional status was associated with levels of retained uremic solutes. LIMITATIONS: Small sample size, only cross-sectional associations can be described. CONCLUSIONS: Our results suggest an association between poor nutritional status and impaired olfactory function in patients with ESRD. Additional research is needed to discover the uremic toxins mediating these processes.

Indoxyl sulfate-lowering capacity of oral sorbents affects the prognosis of kidney function and oxidative stress in chronic kidney disease.

OBJECTIVES: Indoxyl sulfate shows nephrotoxicity and is a stimulating factor for progression of chronic kidney disease (CKD). This study was conducted to determine (1) whether the indoxyl sulfate-lowering capacity of oral sorbents (Kremezin [AST-120], Kureha Corporation, Tokyo, Japan; Merckmezin, Merck Hoei Ltd., Osaka, Japan) affects the prognosis of kidney function in CKD, and (2) whether oral sorbents reduce the markers of oxidative stress. METHODS: Rats with CKD were produced by 4/5 nephrectomy and were randomized into 3 groups: control rats, Merckmezin-treated rats, and Kremezin-treated rats. Kremezin and Merckmezin were administered to rats at a dose of 4 g/kg with powder chow for 16 weeks, whereas powder chow alone was administered to control rats. RESULTS: Administration of Kremezin significantly decreased serum and urine levels of indoxyl sulfate and serum creatinine and significantly increased creatinine clearance as compared with control values. The change in serum indoxyl sulfate noted from the initial to the final week showed a positive correlation with the change in serum creatinine and a negative correlation with the change in creatinine clearance. Kremezin significantly reduced urine levels of acrolein, a marker of oxidative stress, as compared with control levels. CONCLUSIONS: The indoxyl sulfate-lowering capacity of oral adsorbents affects the prognosis of kidney function in CKD. The more serum indoxyl sulfate is reduced, the better kidney function is preserved. Kremezin alleviates oxidative stress in the kidneys by reducing serum levels of indoxyl sulfate.

Spontaneous variability of pre-dialysis concentrations of uremic toxins over time in stable hemodialysis patients.

BACKGROUND AND AIM: Numerous outcome studies and interventional trials in hemodialysis (HD) patients are based on uremic toxin concentrations determined at one single or a limited number of time points. The reliability of these studies however entirely depends on how representative these cross-sectional concentrations are. We therefore investigated the variability of predialysis concentrations of uremic toxins over time. METHODS: Prospectively collected predialysis serum samples of the midweek session of week 0, 1, 2, 3, 4, 8, 12, and 16 were analyzed for a panel of uremic toxins in stable chronic HD patients (N = 18) while maintaining dialyzer type and dialysis mode during the study period. RESULTS: Concentrations of the analyzed uremic toxins varied substantially between individuals, but also within stable HD patients (intra-patient variability). For urea, creatinine, beta-2-microglobulin, and some protein-bound uremic toxins, Intra-class Correlation Coefficient (ICC) was higher than 0.7. However, for phosphorus, uric acid, symmetric and asymmetric dimethylarginine, and the protein-bound toxins hippuric acid and indoxyl sulfate, ICC values were below 0.7, implying a concentration variability within the individual patient even exceeding 65% of the observed inter-patient variability. CONCLUSION: Intra-patient variability may affect the interpretation of the association between a single concentration of certain uremic toxins and outcomes. When performing future outcome and interventional studies with uremic toxins other than described here, one should quantify their intra-patient variability and take into account that for solutes with a large intra-patient variability associations could be missed.

Accumulation of indoxyl sulfate in OAT1/3-positive tubular cells in kidneys of patients with chronic renal failure.

Indoxyl sulfate shows nephrotoxicity and is a stimulating factor for progression of chronic renal failure (CRF). Indoxyl sulfate is taken up by renal proximal tubular cells through organic anion transporters 1 and 3 (OAT1/3), and is accumulated in the renal proximal tubular cells of uremic rats. To determine whether indoxyl sulfate is accumulated in human OAT1/3 (hOAT1/3)-positive renal proximal tubular cells, localization of indoxyl sulfate and hOAT1/3 in the kidneys of CRF patients was determined by immunohistochemistry. Kidney samples were obtained by autopsy from 9 CRF patients (mean serum creatinine 4.7 mg/dL, ranging from 2.0 to 14.5 mg/dL) and 9 patients with non-kidney disease (mean serum creatinine 0.6 mg/dL, ranging from 0.4 to 0.9 mg/dL). Immunohistochemistry was performed using antibodies against indoxyl sulfate, hOAT1, and hOAT3. Indoxyl sulfate was localized in the hOAT1- and hOAT3-positive renal tubular cells in the kidneys of CRF patients. The indoxyl sulfate-positive area in the kidneys was markedly increased in the kidneys of CRF patients compared with patients with non-kidney disease. The indoxyl sulfate-positive area was positively correlated with serum creatinine. In conclusion, in CRF patients, indoxyl sulfate is accumulated in the tubular cells with hOAT1 and/or hOAT3 localized at the basolateral membrane. The extent of indoxyl sulfate accumulation in the kidneys is more prominent in those patients with more severe CRF.

Development and validation of a LC-MS/MS method for quantitative analysis of uraemic toxins p-cresol sulphate and indoxyl sulphate in saliva.

p-Cresol sulphate (pCS) and indoxyl sulphate (IS) are uraemic toxins, the concentration of which in serum correlate with the stage of renal failure. The aim of this study was to develop and validate a high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the analysis of pCS and IS in saliva. This is the first time, to our knowledge, that such a method has been developed using saliva. Unstimulated, fasting saliva was collected from healthy volunteers in the morning and pooled for validation assay. The method was validated for linearity, precision, accuracy, stability (freeze/thaw stability, stability in autosampler, short- and long-term stability, stock solution stability), dilution integrity and matrix effect. The analysed validation criteria were fulfilled. No influence of salivary flow (pCS: p=0.678; IS: p=0.238) nor type of swab in the Salivette device was detected. Finally, using the novel validated method, the saliva samples of healthy people (n=70) of various ages were analysed. We observed a tendency for an increase of concentration of toxins in saliva in the elderly. This could be a result of age-related diseases, e.g., diabetes and kidney function decline. We can conclude that the novel LC-MS/MS method can be used for the determination of pCS and IS in human saliva. The results encourage the validation of saliva as a clinical sample for monitoring toxin levels in organisms.

Rifaximin preserves intestinal microbiota balance in patients undergoing allogeneic stem cell transplantation.

Intestinal dysbiosis has been associated with acute gastrointestinal GvHD and poor outcome following allogeneic stem cell transplantation (ASCT). To assess the effect of a switch in 2012 from ciprofloxacin/metronidazole to rifaximin for gut decontamination on intestinal microbiota composition and ASCT outcome, we retrospectively analyzed 394 patients receiving ASCT from September 2008 through June 2015. In 131 and 90 patients, respectively, urinary 3-indoxyl sulfate levels and intestinal enterococcal load were measured before conditioning and weekly within the first 28 days after ASCT. The use of rifaximin correlated with lower enterococcal positivity (6.9 vs 21.9%, P=0.05) and higher urinary 3-indoxyl sulfate concentrations (10.5 vs 4.6 µmoL/mmoL crea, P<0.001) after ASCT. Patients on rifaximin showed lower 1-year transplant-related mortality (P=0.04) and higher overall survival (P=0.008). Treatment of infectious complications with systemic antibiotics did not abrogate the beneficial effects of rifaximin on intestinal microbiota composition in the early course of ASCT and outcome. The data underscore the importance of maintaining a diverse population of symbiotic and mutualistic bacteria in the gut on ASCT outcome.

A new HPLC-ELSD method to quantify indican in Polygonum tinctorium L. and to evaluate beta-glucosidase hydrolysis of indican for indigo production.

A method to quantify the indigo precursor indican (indoxyl-beta-D-glucoside) in Polygonum tinctorium L. has been developed. Plant material was extracted in deionized water, and indican was identified and quantified using high performance liquid chromatography (HPLC) coupled to an evaporative light scattering detector (ELSD). Results confirmed that with this method it is possible to measure indican content in a short time, obtaining reliable and reproducible data. Using this method, leaf indican content was quantified every 15 days during the growing season (from May to October) in P. tinctorium crops grown in a field experiment in Central Italy. Results showed that indican increased along the growing season until flowering and was positively affected by photosynthetic active radiation (PAR). Indican is naturally hydrolyzed by native beta-glucosidase to indoxyl and glucose, the indoxyl yielding indigo. The activity of two enzymes, sweet almond beta-glucosidase and Novarom G preparation, were compared with P. tinctorium native beta-glucosidase to evaluate indigo production. Results showed that the ability to promote indigo formation increased as follows: almond beta-glucosidase

Environmental factors affecting productivity, indican content, and indigo yield in Polygonum tinctorium Ait., a subtropical crop grown under temperate conditions.

Polygonum tinctorium Ait. is a herbaceous subtropical annual plant, belonging to the family Polygonaceae. Within the cells of its leaves P. tinctorium accumulates large amounts of a colorless glycoside, indican (indoxyl beta-d-glucoside), from which the blue dye indigo is synthesized. P. tinctorium is well-known in Japan, where it had been cultivated to produce natural indigo for textile dyeing, whereas it represents a potentially interesting new crop in Europe. To better understand the effects of environmental parameters on P. tinctorium crop production and indigo yield, field experiments were carried out in central Italy under temperate climate. Three lines were tested during the 2001 and 2002 growing seasons, and plant/leaf yields as well as indican contents were evaluated. The results showed that P. tinctorium grown in temperate climate conditions can be harvested three times a year. Yields of 82 and 120 t ha(-1) of fresh plant yield were obtained in 2001 and 2002, respectively. The contrasting weather conditions between the two years significantly affected biomass production, which was higher in the 2002 season, characterized by wet weather conditions. The cycle length from sowing to the last harvest was accomplished in 229-238 days when plants had accumulated 2017-2018 degrees C. Green leaves accounted for 40-45% by weight of fresh plant tissue and contained 11-20 g kg(-1) indican. The three lines did not significantly differ in the main productive parameters or in fresh leaf indican content (14.1 g kg(-1) mean value). Photosynthetic active radiation influences indican leaf production according to the model y = 0.0004x + 8.566 (P < 0.01, correlation coefficient = 0.818). Indican content ranged from 12 to 25 g kg(-1) of fresh leaves with PAR daily values from 10000 to 40000 mEinstein m(-2) (recorded in May and at the end of July-beginning of August, respectively). The results indicate that in nonlimiting rainfall conditions a very high indican content and a potentially high indigo yield can be obtained by cultivating P. tinctorium in this pioneer geographical area.

Quantitative 1H-NMR analysis of amniotic fluid.

Ten amniotic fluid samples (36-38 weeks gestation) are analysed by NMR spectroscopy. Of the species identified in the spectra, valine (mean 198 microM: SEM 57 microM), lactate (9.73 mM; 2.05 mM), alanine (689 microM: 115 microM), acetate (6.87 mM: 1.54 mM), citrate (363 microM: 59 microM), glucose (4.54 mM: 1.28 mM) indoxyl-sulphate (n = 4,270 microM), histidine (n = 6, 125 microM: 31 microM) and formate (n = 4, 92 microM) are quantified using standard addition. The factors governing the detection limits and lowest quantifiable amounts are discussed as are the extension of the work into in vivo magnetic resonance spectroscopy (MRS) in the clinic.