Marco Bagnati

Alterations of Na+ homeostasis in hepatocyte reoxygenation injury

Reperfusion injury represents an important cause of primary graft non-function during liver transplantation. However, the mechanism responsible for cellular damage during reoxygenation has not yet been completely understood. We have investigated whether changes in intracellular Na(+) distribution might contribute to cause hepatocyte damage during reoxygenation buffer after 24 h of cold storage. Hepatocyte reoxygenation resulted in a rapid increase in cellular Na(+) content that was associated with cytotoxicity. Na(+) accumulation and hepatocyte death were prevented by the omission of Na(+) from the incubation medium, but not by the addition of antioxidants. Blocking Na(+)/H(+) exchanger and Na(+)/HCO(3)(-) co-transporter by, respectively, 5-(N,N-dimethyl)-amiloride or omitting HCO(3)(-) from the reoxygenation medium significantly decreased Na(+) overload and cytotoxicity. Stimulation of ATP re-synthesis by the addition of fructose also lowered Na(+) accumulation and cell death during reoxygenation. A significant protection against Na(+)-mediated reoxygenation injury was evident in hepatocytes maintained in an acidic buffer (pH 6.5) or in the presence of glycine. The cytoprotective action of glycine or of the acidic buffer was reverted by promoting Na(+) influx with the Na(+)/H(+) ionophore monensin. Altogether, these results suggest that Na(+) accumulation during the early phases of reoxygenation might contribute to liver graft reperfusion injury.

Different mechanisms are progressively recruited to promote Cu(II) reduction by isolated human low-density lipoprotein undergoing oxidation.

The kinetics of Cu(II) reduction and its relationship to the process of low density lipoprotein (LDL) oxidation were investigated in isolated human LDL incubated with CuSO4 by using the Cu(I) chelator and indicator dye bathocuproine disulfonate (BC). The inclusion of BC in the incubation medium containing isolated LDL and different concentrations of CuSO4 revealed a biphasic kinetics of Cu(II) reduction consisting of an early phase followed by a plateau phase and a subsequent extensive reduction phase. The amount of Cu(I) formed during the early phase, as well as the rate of its generation, were strictly dependent on both the level of Cu(II) available (saturation was observed at 20 and 50 microM CuSO4) and the concentration of alpha-tocopherol within native LDL particles. Artificial enrichment of LDL with different concentrations of alpha-tocopherol led to a parallel increase of both the amount of Cu(II) reduced and the rate of reduction. The late phase of Cu(II) reduction was strictly related to the availability of copper but was largely independent from alpha-tocopherol. Neither the amount of Cu(I) generated nor the rate of generation were saturated at concentrations of copper up to 100 microM. Comparable results were obtained by adding BC at different time-points to the LDL-copper mixture, in order to measure at the same time-points both the true rate of Cu(II) reduction and the generation of TBARS during the dynamic process of LDL oxidation. The rate of Cu(II) reduction was already high during the lag-phase of the LDL oxidation profile and progressively decreased as alpha-tocopherol concentration decreased. The subsequent increase in the rate of Cu(II) reduction paralleled the formation of TBARS during the extensive LDL oxidation phase. These results suggest that different mechanisms of Cu(II) reduction, namely alpha-tocopherol-dependent and independent (likely lipid peroxide-dependent), are progressively recruited during copper-promoted LDL oxidation.

Vitamin D Deficiency and Glycemic Status in Children and Adolescents with Type 1 Diabetes Mellitus

Background Vitamin D (25OHD) effects on glycemic control are unclear in children and adolescents with type 1 diabetes. Aims of this study were to investigate 25OHD status among children with T1DM and its relationship with insulin sensitivity and glycemic status. Subjects and Methods A cross sectional study was carried out between 2008–2014. A total of 141 patients had a T1DM >12 months diagnosis and were enrolled in the present study. Of these 35 (24.8%) were migrants and 106 (75.2%) Italians (T2). We retrospectively analyzed data at the onset of the disease (T0)(64 subjects) and 12–24 months before the last visit (T1,124 subjects). Fasting glucose, glycated hemoglobin (HbA1c), 25OHD levels and daily insulin requirement were evaluated and Cholecalciferol 1000 IU/day supplementation for the management of vitamin D insufficiency (<75 nmol/L) was systematically added. Results A generalized 25OHD insufficiency was found at each study time, particularly in migrants. At T0, the 25OHD levels were inversely related to diabetic keto-acidosis (DKA) severity (p<0.05). At T1 and T2, subjects with 25OHD ≤25nmol/L (10 ng/mL) showed higher daily insulin requirement (p<0.05) and HbA1c values (p<0.01) than others vitamin D status. The 25OHD levels were negatively related with HbA1c (p<0.001) and daily insulin dose (p<0.05) during follow up. There was a significant difference in 25OHD (p<0.01) between subjects with different metabolic control (HbA1c <7.5%,7.5–8%,>8%), both at T1 and T2. In supplemented subjects, we found a significant increase in 25OHD levels (p<0.0001) and decrease of HbA1c (p<0.001) between T1 and T2, but this was not significant in the migrants subgroup. Multivariate regression analysis showed a link between HbA1c and 25OHD levels (p<0.001). Conclusions Children with T1DM show a generalized 25OHD deficiency that impact on metabolic status and glycemic homeostasis. Vitamin D supplementation improves glycemic control and should be considered as an additional therapy.

Measurement of total antioxidant capacity of human plasma: Setting and validation of the CUPRAC–BCS method on routine apparatus ADVIA 2400

BACKGROUND Quantification of Total Antioxidant Capacity (TAC) of human plasma is an important clinical target, since many diseases are suspected to be related with oxidative stress. The CUPRAC-BCS (BCS=Bathocuproinedisulfonic acid) method was chosen since it works using the photometric principle, with stable and inexpensive reagents and at physiological pH. METHODS The method is based on the complex equilibria between Cu(II)-BCS (reagent) and Cu(I)-BCS. Cu(I)-BCS complex is formed by reducing ability of the plasma redox active substances. The photometric signal is achieved at 478 nm and calibration is performed using urate as a reference substance. RESULTS Linearity, linear working range, sensitivity, precision, LoD, LoQ, selectivity and robustness have been considered to validate the method. Absorbance at 478 nm was found linear from 0.0025 up to 2.0 mmol L(-1) of urate reference solution. Precision was evaluated as within-day repeatability, Sr=4 µmol L(-1), and intermediate-precision, SI(T)=15 µmol L(-1). LoD and LoQ, resulted equal to 7.0 µmol L(-1) and 21 µmol L(-1) respectively while robustness was tested having care for pH variation during PBS buffer preparation. Tests on plasma (80 samples) and on human cerebrospinal fluid (30 samples) were conducted and discussed. CONCLUSIONS By the analytical point of view, the photometric method was found to be simple, rapid, widely linear and reliable for the routine analysis of a clinical laboratory. By the clinical point of view, the method response is suitable for the study of chemical plasma quantities related to redox reactivity.

Evaluation of amoxicillin plasma and tissue levels in pediatric patients undergoing tonsillectomy

OBJECTIVE Tonsillectomy is the most common surgery performed in the pediatric and young adult populations. Although recent guidelines based on meta-analysis suggest that perioperative chemoprophylaxis plays a role in reducing bacteraemia-related post-tonsillectomy complications, there is no evidence or agreement upon which specific antibiotic, dosage or administration route should be preferred. Since few previous studies have assessed the effectiveness of prophylaxis by direct measurement of antibiotic levels both in plasma and tissue, we designed an experimental study to quantitatively evaluate amoxicillin concentrations in children ready for tonsillectomy and compare these plasma and tissue levels with the Minimal Inhibitory Concentrations (MIC) of the bacteria more commonly involved in the upper airway infections. METHODS Thirty-three pediatric patients under 14 years of age (median 5.0, IQR 4-7, range 3-11; M:F 18:15) with recurrent tonsillitis were treated with 3 doses (established on patients weight) of amoxicillin-clavulanic acid given orally the day before plus a further dose 2h before tonsillectomy. Amoxicillin concentrations on both homogenated tonsillar cores and plasma were measured by HPLC-UV. Bacterial epidemiology and susceptibility were derived respectively from survey data collected by Microbiology Unit and MIC according to the National Committee for Clinical Laboratory Standards (NCCLS). RESULTS Median plasma and tissue amoxicillin concentrations were respectively 4.7 microg/ml (IQR 2.1-8.0; min-max 0.4-14.3) and 1.1 microg/g (IQR 0.4-2.1; min-max 0.4-12.9), considerably below the selected target MIC of pathogens involved in the upper respiratory tract infections (S. aureus, H. influenzae, M. catarrhalis). 20 Children showed undetectable amoxicillin levels in one or both tonsils. Interestingly, 7 out of these patients (35%) had plasma concentrations higher than the target MIC (8 microg/ml). No patient displayed plasma concentrations under the limit of sensitivity of the method. Poor core-plasma and left-right core correlation was observed among patients, suggesting that fibrosis developed after recurrent tonsillitis may hamper antibiotic penetration. CONCLUSIONS Based upon direct measurement of antibiotic levels in plasma and tissue, this study suggests that a revision of the oral prophylaxis in children is required in order to reduce microbial charge in the operative field and accordingly improve the recovery after tonsillectomy.

Accidental digitoxin intoxication: an interplay between laboratory and clinical medicine

Introduction: Two Italian adults arrived at the Emergency Department referring diarrhea, nausea and vomiting for 4 days; weakness, fatigue and visual hallucinations were also complained of. Patients reported the ingestion of some leaves of a plant, which they supposed to be “donkey ears”, a week before. Physical examination showed hypotension and bradycardia and ECG examination disclosed sinus rhythm and repolarization abnormalities (scooping of the ST-T complex) in both patients and a 2:1 AV block in the man. Materials and methods: Digoxin concentration was evaluated twice for each patient (at the admission and after 4 hours) by the automated immunoassay system ADVIA Centaur®. Digitoxin concentration was evaluated by liquid chromatography-mass spectrometry (LC-MS/MS). Results: Despite clinical picture was suggestive of digitalis intoxication, digoxin levels were undetectable. Due to the more severe clinical picture, the male patient was treated with anti-digoxin antibodies (Digifab®) achieving a good clinical improvement and remission of the AV block within two hours. Initial diagnosis was confirmed by LC-MS/MS showing high digitoxin concentrations, but digoxin was undetectable. Patients remained stable and 48 hours later were discharged from the hospital. Conclusion: Whereas digoxin determination frequently relies on monoclonal antibodies which do not cross-react to digitoxin, polyclonal antibodies constituting Digifab® recognize a large spectrum of cardiac glycosides, including digitoxin. This report emphasizes the primary role of the clinical approach to patients in the emergency setting and how an active communication and a continuous sharing of professional experiences between Laboratory and Clinicians ensure an early and correct diagnosis.

False negativity to carbohydrate-deficient transferrin and drugs: a clinical case

Introduction: In this work we report on the possible effect of the medical therapy on CDT concentration in a chronic alcohol abuser, with known medical history (July 2007 – April 2012) and alcohol abuse confirmed by relatives. Case history: At the end of 2007, patient displayed the following laboratory results: AST 137 U/L, ALT 120 U/L, GGT 434 U/L, MCV 101 fL and CDT 3.3%. On December 2007, after double coronary artery bypass surgery, he began a pharmacological treatment with amlodipine, perindopril, atorvastatin, isosorbide mononitrate, carvedilol, ticlopidine and pantoprazole. In the next months, until may 2011, the patient resumed alcohol abuse, as confirmed by relatives; however, CDT values were repeatedly found negative (0.8% and 1.1%) despite elevated transaminases and GGT, concurrent elevated ethyl glucuronide concentration (> 50 mg/L) and blood alcohol concentration (> 1 g/L). Alcohol consumption still continued despite increasing disulfiram doses ordered by an Alcohol Rehab Center. On May 2011, the patient was transferred to a private medical center where he currently lives. Conclusions: This study suggests the possibility that a medical therapy including different drugs may hamper the identification of chronic alcohol abusers by CDT.

Recognizing purple bag syndrome at first look

Purple urine bag syndrome is a clinical entity first described in 1978. Its typical discoloration is worrying for clinicians. In the past, these patients sometimes reached the emergency unit only because of this exceptional worrying urinary sign and underwent invasive diagnostic examinations including cystoscopy, without any abnormal finding. It is now clear that this astonishing phenomenon of double discoloration of the urine, appearing purple in the bag and dark blue in the test tube, results from the formation of 2 different pigments (indirubin and indigo) in very alkaline urines due to enzymes produced by gram-negative bacteria, such as indoxyl phosphatase/sulfatase, which can convert urinary metabolites of dietary tryptophan. Practicing physicians should identify purple urine bag syndrome as a usually benign medical condition diagnosed in asymptomatic patients, which only requires treatment of bacteriuria with antibiotics, prevention of constipation, substitution of catheter and acidification of the urine. After these measures, urine typically returns to its normal color.

Purifying Capacity of a New Membrane Characterized by a Medium Cut-Off and High Performance

Introduction: The adsorbing capacity of membranes can be considered the third dimension of the depurative action of dialysis: diffusion, convection and adsorption. Some metabolites are almost totally bound to albumin, so, regardless of their molecular weight, removal with low flux or high flux dialysis membranes becomes difficult if not impossible. The aim of our work was to evaluate the adsorptive efficacy as well as the depurative of a new membrane with a medium cut-off: Theranova 400, PAES / PVP membrane with surface 1.7 m2 Baxter. Materials and methods: Four patients in chronic three-weekly dialysis were evaluated for over 6 months, treated with the Theranova 400 dialyzer filter, for 6 dialysis bicarbonate sessions. In every piece, blood tests were performed at the beginning and end of dialysis, for the first 3 dialysis and at the third dialysis of the second week, to evaluate besides the purification of the main standard parameters, also the removal of medium molecular weight solutes such as myoglobin, beta2 microglobulin and solutes such as indoxyl sulfate (F IXS) and pcresol (F PCS) which represent less than 10% of the free fraction being bound to albumin for more than 90% of their total concentration (T IXS and T PCS). IXS and PCS were also measured at the biginning of the third dialysis session of the week , in other 130 ESRD patients treated with various membranes . The statistical differences, before and after dialysis, were calculated with the Student’s t test for paired data. For the comparison of the values between Theranova and other membranes the Mann Whitney U Test was used for nonparametric data. Results: At the end of the 6 4-h hemodialysis sessions there was a significant decrease in both the low molecular weight parameters with a good KT/V and a urea clearance close to 300 ml/min. Mean MW concentrations were reduced by 50-75%, while F IXS, T IXS, F PCS and T PCS were reduced by more than 20%. The predialytic values of the last dialysis of the week tended to be higher and in some statistically significant in all the other membranes compared to Theranova. Conclusions: In addition to the high diffusional purifying capacity, the membrane shows a marked adsorptive capacity demonstrated by the effective removal of metabolites linked to albumin and therefore more difficult to remove. It will be interesting to follow in time the adsorptive capacity of this membrane and its clinical implication, especially in comparisons of cardiovascular and atherosclerotic diseases that appear to be related to the concentrations of these metabolites, practically impossible to remove with standard membranes.

Short-Term Effects of Pre/Probiotics on P-Cresol and Indoxyl-Sulphate Serum Concentrations During the Various Stages of Chronic Kidney Disease

Background: The uremic syndrome is provoked by a progressive number of compounds that are normally excreted by kidneys in healthy individuals. Indoxylsulphate (IXS) and p-cresylsulphate (PCS), have been found increased in subjects with end stage renal disease (ESRD) creating great harm to biological systems; these uremic toxins come from the intestinal bacterial fermentation of the proteins. The aim of our study is to evaluate the short-term effects after an administration of pre / probiotics in CKD patients, regarding the production and then the serum concentrations of free IXS and PCS (i.e. non-protein bound fraction) and total IXS and PCS ( i.e. sum of unbound and protein bound fraction). Methods: In our study , 26 patients with CKD stage 2-5 associated with hypertension and / or diabetes mellitus type 2 were enrolled, and administered with 2 g/day dose of pre-probiotics for four months: mixed oligofructose (prebiotic component) + Lactobacillus acidophilus and Bifi dobacterium longum (probiotic component). In all patients, at the beginning of the study, kidney function tests, glucose metabolism, PTH and blood uric acid were evaluated. Free and total PCS and IXS were also measured. 20 control subjects with normal renal function were considered in relation to the same parameters. After 4 months 19 patients were re-evaluated in relation to the same parameters. Statistical differences were studied using the Student-t paired and unpaired tests. Results: The baseline values of IXS and PCS of the 26 patients were signifi cantly higher compared with the normal subjects and importantly increased with the transition to the higher stage of CKD. The values in stage 2 3 CKD were signifi cantly lower in respect to stage 4 – 5 CKD. Higher mean values of IXS and PCS in 12 diabetic subjects were highlighted, although not statistically signifi cant compared to 14 hypertensive non-diabetic patients. The data after the use of pre probiotics in 19 patients that completed the treatment protocol (5 patients were out of the study for non-compliance of the processing and 2 patients for dialysis entrance), showed increased concentrations of free and total IXS and PCS. Considering renal function, the use of pre probiotics increased the concentration of free and total PCS and IXS in all conditions, while remaining signifi cantly higher in patients at stage 4-5 rather than in the ones at the stage 2-3. The use of pre probiotics increased the IXS and PCS serum concentrations, remaining signifi cantly higher in diabetics rather than in hypertensive patients. In all periods, both baseline and after the uptake of pre-probiotics, the other measured parameters didn’t change except serum PTH that decreased signifi cantly and Calcium increased even if not signifi cantly Conclusions: In conclusion IXS and PCS can be considered as kidney function markers as well as have systemic toxic effects. Diabetes seems to increase the concentration of the two metabolites. The use of pre-probiotics should be started in the early stages of kidney failure and certainly for periods longer than four months. Pre-probiotics could aid in preventing renal osteodystrophy. Research Article Short-Term Effects of Pre/Probiotics on P-Cresol and Indoxyl-Sulphate Serum Concentrations During the Various Stages of Chronic Kidney Disease Filomena Panza2, Diletta Duranti1, Ralli Chiara2, Matteo Basile1, Marco Bagnati1, Giorgio Bellomo1 and Ennio Duranti2* 1Department of Pathology and Clinical Laboratories AUO Novara 2UOC Nephrology and Dialysis Unit, Hospital of Arezzo Dates: Received: 14 March, 2017; Accepted: 29 April, 2017; Published: 03 May, 2017 *Corresponding author: Ennio Duranti, UOC Nephrology and Dialysis Unit, Hospital of Arezzo, Italy. E-mail: https://www.peertechz.com