Lucia Grumetto

Measurement of bisphenol A and bisphenol B levels in human blood sera from healthy and endometriotic women

A sensitive HPLC method with fluorescence detection was developed for the determination of bisphenol A (BPA) and bisphenol B (BPB) in human blood serum. The detection limits of the method were 0.18 and 0.20 ng/mL for BPA and BPB, respectively. A single-step liquid-liquid extraction was used for the pre-treatment of serum samples. The recoveries of BPA and BPB spiked to sera were 85.6 and 87.7%, respectively. The analyses of sera from both healthy and endometriotic women emphasized the absence of bisphenols in all the control cases (11 women), whereas BPA was found in 30 sera (51.7%) and BPB was found in 16 sera (27.6%) in the group of 58 patients with endometriosis; in nine of such sera BPA and BPB were present simultaneously. Only relatively to the sera quantitated, BPA concentrations ranged from 0.79 to 7.12 ng/mL (mean concentration 2.91 +/- 1.74 ng/mL), whereas BPB concentrations ranged from 0.88 to 11.94 ng/mL (mean concentration 5.15 +/- 4.16 ng/mL). Therefore, the presence of at least one of the two bisphenols was verified in a percentage as high as 63.8% in the sera from endometriotic women, suggesting the existence of a relationship between endometriosis and BPA and/or BPB exposure. Indeed, it is well known that bisphenols can work as xenoestrogens, owing to their structural similarity to natural and synthetic estrogens (e.g. estradiol and dietilstilbestrol). However, further studies are necessary to confirm this hypothesis and to assess the actual dose at which exposures to bisphenols are able to increase the sensitivity of the endometriotic cells to estradiol.

Determination of Bisphenol A and Bisphenol B Residues in Canned Peeled Tomatoes by Reversed-Phase Liquid Chromatography

Bisphenol A (BPA) and bisphenol B (BPB) concentrations were determined in peeled canned tomatoes of different brands bought in Italian supermarkets. Tomato samples analyzed were packaged in cans coated with either epoxyphenolic lacquer or low BADGE enamel. A solid phase extraction (SPE) was performed on C-18 Strata E cartridge followed by a step on Florisil cartridge. Detection and quantitation were performed by a reversed phase high-performance liquid chromatography (RP-HPLC) method with both UV and fluorescence detection (FD). On the total of 42 tested tomato samples, BPA was detected in 22 samples (52.4%), while BPB was detected in 9 samples (21.4%). BPA and BPB were simultaneously present in 8 of the analyzed samples. The levels of BPA found in this study are much lower than the European Union migration limits of 3 mg/kg food and reasonably unable to produce a daily intake exceeding the limit of 0.05 mg/kg body weight, established by European Food Safety Authority.

Effect of short-term synbiotic treatment on plasma p-cresol levels in patients with chronic renal failure: A randomized clinical trial

BACKGROUND AND AIMS In patients with chronic kidney disease (CKD), alterations in gut microbiome are posited to be responsible for gastrointestinal symptoms and generation of p-cresol, a uremic toxin that has been associated with CKD progression and cardiovascular mortality. This pilot study investigated whether Probinul-neutro®, a synbiotic that normalizes intestinal microflora, may lower plasma p-cresol concentrations and reduce gastrointestinal symptoms in non-dialyzed CKD patients. METHODS AND RESULTS This was a double-blind, randomized placebo-controlled trial. Thirty patients on 3-4 CKD stages were randomized to receive either Probinul neutro® or placebo for 4 weeks. Total plasma p-cresol concentration was assessed at baseline, and 15 and 30 days after treatment start. At the same study times, ease and frequency of defecation, upper and lower abdominal pain, stool shape, borborygmi, and flatus were quantified by subjective assessment questionnaires. Compared to baseline total plasma p-cresol median concentrations on 15th and 30th day were significantly lower in patients receiving Probinul-neutro® (2.31 and 0.78 vs. 3.05 μg/ml, p < 0.05; n = 18); no changes of plasma p-cresol concentrations were recorded in placebo-treated patients. No significant changes in gastrointestinal symptoms were observed during the study both in Probinul-neutro®-treated and placebo-treated patients. CONCLUSION Probinul-neutro® lowered total plasma p-cresol concentrations but did not ameliorate gastrointestinal symptoms in non-dialyzed CKD patients. Because high plasma concentrations of p-cresol in early phases of CKD are predictive of progression to end-stage renal disease, the results of our study suggest that synbiotics deserve attention as possible tools to delay CKD progression towards end-stage renal disease (ESRD). CLINICALTRIALSGOV IDENTIFIER NCT02008331.

Monitoring of bisphenol A and bisphenol S in thermal paper receipts from the Italian market and estimated transdermal human intake: A pilot study.

Bisphenol A (BPA), a synthetic xenoestrogen widely used in various industrial fields, can be present, in its un-reacted form, as an additive in thermal paper. BPA is virtually ubiquitous in industrialized societies and humans are exposed to this chemical via dietary and non-dietary sources. Since in 2015 European Food Safety Authority (EFSA) indicated that thermal paper is the second source of BPA exposure after the food chain, some suppliers replaced BPA with its analogue Bisphenol S (BPS), speculatively supposed to be safer. In this work BPA and BPS concentration levels were determined in thermal paper receipts collected in Italy from 50 different sources by liquid chromatography coupled to tandem fluorescence and ultraviolet detection. BPA was found in 44 samples at mean concentration of 107.47μg/100mg of paper (from below Limits of Quantification (LOQ) to 1533.733μg/100mg of paper). BPS was found in 31 samples at mean concentration of 41.97μg/100mg of paper (from below the LOQ to 357.989μg/100mg of paper). 26 samples were positive to both BPA and BPS. The estimate daily intake (EDI) values of BPA and BPS occurring through dermal absorption were calculated for 70kg body weight individuals. For general population, they were 0.0625μg/day for BPA and 0.0244μg/day for BPS, based on the mean content of bisphenols found. For occupationally exposed individuals, they were 66.8μg/day for BPA and 15.6μg/day for BPS, based on the worst scenario. Such levels would produce a dermal intake below the Tolerable Day Intake established by EFSA (4μg/kg·bw/day); nevertheless, the occurrence of co-exposure to dietary and non-dietary sources should be considered in the health risk assessment, mainly for people frequently exposed to thermal paper contact for occupational reason.

Plasma p-Cresol Lowering Effect of Sevelamer in Peritoneal Dialysis Patients: Evidence from a Cross-Sectional Observational Study

p-Cresol is a by-product of the metabolism of aromatic aminoacid operated by resident intestinal bacteria. In patients with chronic kidney disease, the accumulation of p-cresol and of its metabolite p-cresyl-sulphate causes endothelial dysfunction and ultimately increases the cardiovascular risk of these patients. Therapeutic strategies to reduce plasma p-cresol levels are highly demanded but not available yet. Because it has been reported that the phosphate binder sevelamer sequesters p-cresol in vitro we hypothesized that it could do so also in peritoneal dialysis patients. To explore this hypothesis we measured total cresol plasma concentrations in 57 patients with end-stage renal disease on peritoneal dialysis, 29 receiving sevelamer for the treatment of hyperphosphatemia and 28 patients not assuming this drug. Among the patients not assuming sevelamer, 16 were treated with lanthanum whereas the remaining 12 received no drug because they were not hyperphosphatemic. Patients receiving sevelamer had plasma p-cresol and serum high sensitivity C-reactive protein concentrations significantly lower than those receiving lanthanum or no drug. Conversely, no difference was observed among the different groups either in residual glomerular filtration rate, total weekly dialysis dose, total clearance, urine volume, protein catabolic rate, serum albumin or serum phosphate levels. Multiple linear regression analysis showed that none of these variables predicted plasma p-cresol concentrations that, instead, negatively correlated with the use of sevelamer. These results suggest that sevelamer could be an effective strategy to lower p-cresol circulating levels in peritoneal dialysis patients in which it could also favorably affect cardiovascular risk because of its anti-inflammatory effect.

Determination of five bisphenols in commercial milk samples by liquid chromatography coupled to fluorescence detection.

The presence of five bisphenols, i.e., bisphenol F, bisphenol A, bisphenol B, bisphenol F diglycidyl ether, and bisphenol A diglycidyl ether, was monitored in commercial milk packed in plastic bottles marketed in Italy. The new validated method includes a solid-phase extraction procedure followed by liquid chromatography with fluorescence detection. All positive results were confirmed by liquid chromatography-tandem mass spectrometry analysis. The limits of detection and quantification and the recovery percentages indicated that the method is suitable for detecting bisphenols in milk at concentrations far below the legal limits. Of 68 commercial milk samples analyzed, no bisphenol was found in 27 samples (39.7%), and 41 samples (60.3%) contained one or more bisphenols. The bisphenol most frequently found was bisphenol F (36 samples, 52.9%) followed by bisphenol A (20 samples, 29.4%) and bisphenol B (6 samples, 8.8%). Taking into consideration the limits of detection, no sample contained either bisphenol F diglycidyl ether or bisphenol A diglycidyl ether.

Enantioselective retention of β-blocking agents on human serum albumin and α1-acid glycoprotein HPLC columns: Relationships with different scales of lipophilicity

The enantioselective retention of thirteen beta-blockers on HPLC stationary phases supporting human serum albumin (HSA) or alpha(1)-acid glycoprotein (AGP) was investigated. Eight beta-blockers were enantiomerically resolved on the AGP column whereas only four beta-blockers were resolved on the HSA column. Moreover, interactions between beta-blockers and AGP were much stronger than those with HSA. Retention values on both HSA and AGP for less retained enantiomers related well with various lipophilicity parameters, with the best relationships found with log k(w)(IAM) values obtained on HPLC stationary phases supporting phospholipids, i.e. the so-called Immobilized Artificial Membrane (IAM). Differently from n-octanol lipophilicity values, these values encode both lipophilic. Electrostatic intermolecular recognition forces which may be involved in the interaction between ionized analytes, such as beta-blockers, and proteins. However, their effectiveness to describe non-specific interactions with serum-proteins for other classes of drugs needs further investigations. Analyses performed on AGP with eluent containing dimethyloctylamine (DMOA) as the displacer demonstrated that enantioselective sites bind to both (-)-forms and (+)-forms, but the binding to (-)-forms is stronger. The enantiomer competition to bind to a same site may be relevant from a pharmacokinetic point of view when racemic mixtures are administered. Finally, in contrast to previously reported data in the literature, we found that AGP can bind enantioselectively not only the more lipophilic congeners but also the less lipophilic ones.

Evidence That p-Cresol and IL-6 Are Adsorbed by the HFR Cartridge: Towards a New Strategy to Decrease Systemic Inflammation in Dialyzed Patients?

Introduction Hemodialysis (HD) and hemodiafiltration clear only with a low efficiency the plasma from interleukin-6 and p-cresol, two protein-bound uremic toxins associated with high cardiovascular risk in end stage renal disease. HFR Supra is a double-chamber hemodiafiltration system in which the ultrafiltrate returns to the patient after its regeneration through a resin cartridge that binds hydrophobic and protein-bound solutes. In the present study, we evaluated whether the HFR cartridge can also bind total p-cresol and IL-6 and remove them from the ultrafiltrate. Methods We compared the levels of IL-6 and p-cresol in ultrafiltrate samples collected at the inlet (UFin) and at the outlet (UFout) of the cartridge at the start or at the end of a 240 min HFR session in 12 inflamed chronic HD patients. The pro-inflammatory activity of the ultrafiltrate samples was also determined by evaluating the changes that they induced in IL-6 mRNA expression and protein release in peripheral blood mononuclear cells from 12 healthy volunteers. IL-6 and p-cresol circulating levels were also assessed in peripheral plasma blood samples collected before and after HFR and, for comparison, a control HD. Results p-Cresol and IL-6 were lower in UFout than in UFin both at the start and at the end of the HFR session, suggesting that they were retained by the cartridge. IL-6 mRNA expression and release were lower in PBMC incubated with UFout collected at the end than with UFin collected at the start of HFR, suggesting that passage through the cartridge reduced UF pro-inflammatory activity. Plasma total p-cresol decreased by about 53% after HFR, and 37% after HD. IL-6 circulating values were unmodified by either these dialysis procedures. Conclusions This study shows that the HFR-Supra cartridge retains total p-cresol and IL-6 in the ultrafiltrate and lowers plasma total p cresol but not IL-6 levels. Trial Registration ClinicalTrials.gov NCT01865773

Polar interactions drug/phospholipids estimated by IAM-HPLC vs cultured cell line passage data: Their relationships and comparison of their effectiveness in predicting drug human intestinal absorption

The relationships between data of passage through Caco-2 cultured cell lines (log Papp), taken from the literature, for 38 structurally unrelated compounds and both n-octanol lipophilicity parameters (log P(N) and log D(7.4)) and phospholipid affinity indexes were investigated. Phospholipid affinity(log k W(IAM)) was experimentally determined by HPLC on two different phospholipid stationary phases and the polar/electrostatic interaction component drug/phospholipids (Δ log k W(IAM)) was calculated according to a method we previously proposed. Log Papp moderately related to lipophilicity values measured at pH 7.4 (log D(7.4)), according to a parabolic pattern, but poorly related with log k W(IAM). Furthermore, a significant inverse linear relationship with Δ l og k W(IAM) values was only observed for the analytes with m.w. >300 Da, for which paracellular diffusion can be considered a minor transport route in vivo. Indeed, it has been reported that Caco-2 passage data also encode secondary passage mechanisms, which participate in a different extent to the jejunal absorption in vivo and cannot be directly equated to the corresponding human in situ log Peff values, unless a normalization is performed. In an attempt to elucidate this issue, 47 structurally unrelated compounds whose cultured cell line passage data were corrected for the effects of the aqueous boundary layer and paracellular permeability, so as to express transcellular intrinsic permeability, log P 0(Caco-2/MDCK), were also considered. Highly significant inverse linear relationships were observed between log P 0(Caco-2/MDCK) and Δlog k W(IAM) values from both IAM.PC.MG (r(2)=0.765) and IAM.PC.DD2 (r(2)=0.806) stationary phases whereas the relationships with either lipophilicity in n-octanol or log k W(IAM) values were very poor. The results of the present study, in complete agreement with those of our recent study on the relationships between jejunal absorption data measured in situ and Δ log k W(IAM) values, confirm the soundness of Δ log k W(IAM) parameters in the prediction of the intestinal absorption of drugs. From a mechanistic point of view, they suggest that the polar/electrostatic forces between drugs and phospholipids play a major role in the passage through biomembranes.

Determination of dimethyl fumarate in desiccant and antimould sachets by reversed-phase liquid chromatography

A method for the determination of dimethyl fumarate (DMF) in desiccant and antimould sachets, employed for protecting consumer products from humidity and mould, has been developed. The method is based on a solid-liquid extraction followed by HPLC-UV analysis. The method was validated with respect to recovery, linearity, limits of detection and quantitation and precision. The recovery was 98%. The correlation coefficient value (r) was equal to 0.94. Both intra- and inter-day precisions were studied at several concentration levels, being satisfactory in all cases (RSD < 5). Limits of detection and quantification values were in the low microgram per gram level, thus allowing the determination of DMF at concentrations below the limit established (0.1 mg/kg) by the recent EU Directive (Decision 2009/251/EC). The proposed procedure was applied for the determination of the target compound in 41 desiccant and antimould samples. DMF was detected in 39.0% of samples and its content in many samples exceeded the legal limits. The results of our analysis highlight the high risk of exposure to this powerful allergic sensitizer for consumers.