Hemodiafiltration with endogenous reinfusion with and without acetate-free dialysis solutions: effect on ESA requirement.

BACKGROUND: Hemofiltrate reinfusion (HFR) is a form of hemodiafiltration (HDF) in which replacement fluid is constituted by ultrafiltrate from the patient 'regenerated' through a cartridge containing hydrophobic styrene resin. Bicarbonate-based dialysis solutions (DS) used in routine hemodialysis and HDF contain small quantities of acetate (3-5 mM) as a stabilizing agent, one of the major causes of intradialytic hypotension. Acetate-free (AF) DS have recently been made available, substituting acetate with hydrochloric acid. The impact of AF DS during HFR on Hb levels and erythropoietic-stimulating agent (ESA) requirement in chronic dialysis patients was assessed. PATIENTS AND METHODS: After obtaining informed consent, 30 uremic patients treated by standard bicarbonate dialysis (BHD, DS with acetate) were randomized to treatment in 3-month cycles: first AF HFR, followed by HFR with acetate, and again AF HFR. At the beginning and end of each period, Hb and ESA requirements were evaluated. RESULTS: A significant increase in the Hb level was observed throughout all periods of HFR versus BHD (from 11.1 to 11.86 g/dl; p = 0.04), with a significant decrease of ESA requirements from 29,500 to 25,033 IU/month (p = 0.04). CONCLUSION: Regardless of the presence or absence of acetate in DS, HFR per se allows a significant lowering of ESA dosage versus BHD, while at the same time increasing Hb levels. Taking for granted the clinical impact produced, HFR seems to provide a relevant decrease in end-stage renal disease patient costs.

Effect of on-line hemodiafiltration with endogenous reinfusion (HFR) on the calcium-phosphorus metabolism: medium-term effects.

AIM: The purpose of the study was to examine the effect of hemodiafiltration with endogenous reinfusion (HFR) compared to hemodialysis (HD) on 28 uremic patients with secondary hyperparathyroidism (2HPT) but positively selected for good and stable control of phosphatemia in order to evaluate the independent effects of dialysis treatments on bone turnover metabolism. METHODS: The study was divided into 3 periods of observation: a) HD for three months; b) HFR for three months; c) HFR for a further 3 months. We analysed the trend of: whole PTH, 1-84 PTH, 7-84 PTH, alkaline phosphatase and its bone isoenzyme, total and ionised calcium, phosphatemia, dose of phosphate binder agents, beta2-microglobulin, CRP. All the variations found were evaluated through mean values +/- SD, t-tests, multivariate analysis. RESULTS: We observed a deceleration in bone turnover characterized by a reduction of the total and bone alkaline phosphatase (IU/mL) from 92.3 +/- 82.8 and 35.8 +/- 49.8 at the end of HD to 63.4 +/- 23.9 and 16.0 +/- 8.7 at the end of HFR, respectively, and 1-84 PTH from 317.5 +/- 264.6 pg/mL at the end of HD to 287.5 +/- 258.9 pg/mL at the end of the 3rd month of HFR. Beta2-microglobulin was reduced from 32.9 +/- 16.1 mg/L at the end of HD to 26.4 +/- 8.1 mg/L already at the end of the first three months of HFR. CRP was reduced from 2.5 +/- 2.6 mg/dL at the beginning of the study to 1.3 +/- 1.7 mg/dL at the end of HFR. There were no differences with regard to: dialytic efficiency, nutritional status, calcemia, phosphatemia (maintained in the K-DOQI range for the entire duration of the study), also thanks to more careful use of phosphate chelating agents. CONCLUSION: We are of the opinion that HFR - essentially thanks to the use of ultrapure endogenous infusate - induces a deceleration in bone turnover due to 2PHT. In addition, phosphate subtraction in HFR is better compared to HD, thanks to the improvement of the anti-inflammatory conditions by removing the cytokines harmful to bone metabolism and excluding a priori the negative effects related to hyperphosphatemia.

A real-time classification system of thalassemic pathologies based on artificial neural networks.

Thalassemias are pathologies that derive from genetic defects of the globin genes. The most common defects among the population affect the genes that are involved in the synthesis of alpha and beta chains. The main aspects of these pathologies are well explained from a biochemical and genetic point of view. The diagnosis is fundamentally based on hematologic and genetic tests. A genetic analysis is particularly important to determine the carriers of alpha-thalassemia, whose identification by means of the hematologic parameters is more difficult in comparison with heterozygotes for alpha-thalassemia. This work investigates the use of artificial neural networks (ANNs) for the classification of thalassemic pathologies using the hematologic parameters resulting from hemochromocytometric analysis only. Different combinations of ANNs are reported, which allow thalassemia carriers to be discriminated from normals with 94% classification accuracy, 92% sensitivity, and 95% specificity. On the basis of these results, an automated system that allows real-time support for diagnoses is proposed. The automated system interfaces a hemochromo analyzer to a simple PC.

Predilution haemofiltration--the Second Sardinian Multicentre Study: comparisons between haemofiltration and haemodialysis during identical Kt/V and session times in a long-term cross-over study.

BACKGROUND: The potential superiority of various renal replacement treatment modalities consisting largely of convective mass transfer as opposed to primarily diffusive mass transfer, is still a matter of debate. The objective of the present study was to evaluate acute and long-term clinical effects of varying degrees of convection and diffusion in a group of 24 clinically stable patients with end-stage renal disease. METHODS: The patients were prospectively assigned to three consecutive treatment schedules of 6 months each: phase I (HF1) (on-line predilution haemofiltration)-->phase II (HD) (high-flux haemodialysis)-->phase III (HF2; as phase I). We used the AK100/200 ULTRA monitor (Gambro), which prepares ultrapure dialysis fluid for HD and sterile, pyrogen-free substitution solution for HF. The membrane (polyamide), fluid composition, and treatment time were the same on HF and HD. The targeted equilibrated Kt/V was 1.2 for both treatment modes, creating a similar urea clearance. RESULTS: Fifteen patients, mean age 62.8+/-8.4 years, completed the study according to the above conditions. Urea kinetics, nutritional parameters, and dry weight were similar in the three periods. The frequency of intra-treatment episodes of hypotension/patient/month was significantly lower on HF1 (1.24) and HF2 (1.27) than on HD (1.80) (P<0.04). It decreased progressively on HF1, then increased on HD, and decreased again during HF2. Patients had fewer muscular cramps on HF than on HD (P<0.03) and required significantly less saline and plasma expander during HF than HD sessions. The prevalence of inter-treatment symptoms, including fatigue and hypotension, was lower on HF than on HD (score difference P=0.04). Quality of life, determined by the Laupacis method in all three periods, showed a tendency towards improvement during the study, reaching the best values during HF2. CONCLUSIONS: HF has a progressive stabilizing haemodynamic effect, producing a more physiological cardiovascular profile than HD. This long-term effect, observed in stable patients treated under strictly identical conditions, is probably due to the mechanism of convection, and is different from the acute effect observed mainly in unstable patients.

Small ring constrained peptidomimetics. Synthesis of epoxy peptidomimetics, inhibitors of cysteine proteases.

Different dipeptide analogues containing an oxirane ring in the place of the peptidic bond were prepared starting from naturally occurring amino acids. N-Fmoc-amino aldehydes were transformed into the corresponding methoxyvinyl derivatives through a Wittig reaction, and the addition of PhSeCl gave a series of different alpha-phenylselenyl aldehydes. Mukajiama reaction with silylketene acetals gave an intermediate product that was finally transformed into the desired oxiranyl peptidomimetics. Following this strategy we were able to control three new contiguous stereocenters starting from the enantiomerically pure amino acid. The dipeptide analogues could be used in SPPS on a SASRIN resin as the final epoxides were relatively unstable under acidic conditions. Moreover the synthesis of the single dipeptide mimetics was carried out on solid phase to generate a small library of epoxy peptidomimetics. Some of the products prepared in this work resulted as time-dependent reversible inhibitors of cysteine protease.

Dissociation of human alphaB-crystallin aggregates by thiocyanate is structurally and functionally reversible.

Conformational modifications and changes in the aggregation state of human alphaB-crystallin were investigated at different concentrations of SDS, KBr, urea, and NH4SCN and at different temperatures. Intrinsic fluorescence measurements indicated complete and reversible unfolding of the protein at 2 M NH4SCN, whereas the concentration of urea required for complete and irreversible unfolding was 6 M. Gel permeation chromatography indicated almost complete dissociation of the micelle-like aggregate of alphaB-crystallin in 2 M NH4SCN, but only partial dissociation into large-sized aggregates in 6 M urea. Thiocyanate-treated alphaB-crystallin recovered its chaperone-like activity upon dilution of the dissociating agent, whereas the urea-treated protein did not.

Photodegradation of organic waste coupling hydrogenase and titanium dioxide.

In this work the results of the study on the degradation of dairy waste and lactose with use of titanium dioxide (TiO2), as photocatalyst, are presented. The ability of TiO2 to degrade dairy waste is compared either in the presence of molecular oxygen or of a bacterial hydrogenase as electron acceptor. The enzyme-mediated H2 production rates or the O2 consumption rates are used to measure electron donor degradation. The results obtained clearly indicate that dairy waste can be degraded by the TiO2 photocatalyst. Proteins present in the dairy waste have a strong inhibitory effect on the degradation process. Indeed lactose alone can easily be degraded. When the protein extract obtained from the dairy waste was added to the lactose solution, the reaction for both electron acceptors, hydrogenase and oxygen, was inhibited. When the dairy waste solutions were diluted, there was a positive effect on the reaction rate. This was particularly true in the case of hydrogenase, and to a lesser extent in the case of oxygen acceptor. The reduction of the pH from 8 to 6 also increased H2 production when the enzyme was used.

Classification and quantitation of 1H NMR spectra of alditols binary mixtures using artificial neural networks.

A pattern recognition method based on artificial neural networks (ANNs) to analyze and quantify the components of six alditol binary mixtures is presented. This method is suitable to classify the spectra of the 15 mixtures obtained from the six alditols and to produce quantitative estimates of the component concentrations. The system is user-friendly and is helpful in solving the problem of greatly overlapping signals, often encountered in NMR spectroscopy of carbohydrates. A "classification" ANN uses 200 intensity values of the 1H NMR spectrum in the range 3.5-4 ppm. When the correct mixture is identified, the quantification is solved by assigning a specific ANN to each mixture. These ANNs use the same 200 values of the spectrum and output the values of the two concentrations. The error in the ANN responses is studied, and a method is developed to estimate the accuracy in determining the concentrations. The networks' abilities to recognize previously unseen mixtures are tested. When the classification ANN (trained on the 15 binary mixtures) is exposed to complex (i.e., more than binary) mixtures of the six known alditols, it successfully identifies the components if their minimum concentration is 10%. Given the precision of the results and the small number of errors reported, we believe that the method can be used in all fields in which the recognition and quantification of components are necessary.

On-line predilution hemofiltration versus ultrapure high-flux hemodialysis: a multicenter prospective study in 23 patients. Sardinian Collaborative Study Group of On-Line Hemofiltration.

The aims of the present prospective multicenter study were to assess the clinical tolerance and well being, the correlation between nPCr and Kt/V and the pretreatment beta 2-microglobulin level in patients sequentially treated with high-flux dialysis with ultrapure bicarbonate hemodialysis (HD; phase 1) and predilution hemofiltration (HF) with on-line prepared bicarbonate substitution fluid (phase II). The same monitor (Gambro AK 100 ULTRA) and membrane (polyamide) were used. Twenty-three patients, all in a stable clinical condition, entered the study. The treatment was targeted to an equilibrated Kt/V (eqKt/V) of 1.4 for HD and 1.0 for HF. No mortality or relevant morbidity were observed. The number of hypotensive episodes was 1.78 +/- 2.8 per patient and month during HD vs. 1.17 +/- 3.1 during HF (p = 0.003) and the number of the hypertensive episodes 1.28 +/- 2.8 during HD vs. 0.42 +/- 0.8 during HF (p = 0.04). Incidences of arrhythmia, muscular cramps and headache were significantly less frequent during HF. Interdialytic cramps, arthralgia and fatigue were also significantly less frequent during the HF period. The average beta 2-microglobulin level was 27.1 +/- 14.7 mg/dl at the start of the study, 22.9 +/- 4.9 mg/dl at the beginning of phase II and 22.4 +/- 4 mg/dl at the end of phase II (p = 0.01 compared to the start). A significant linear correlation between the normalized protein catabolic rate and eqKt/V was obtained faster during HD than during HF (45 vs. 120 days) indicating that HF affects the nutritional status with mechanisms different from HD. The present study is in agreement with the hypothesis that HF gives and adequate nutritional status with improved clinical stability and well being at a lower Kt/V compared to HD. Both therapies were efficient in controlling the pretreatment beta 2-microglobulin level.

Spectroscopic identification and quantitative analysis of binary mixtures using artificial neural networks.

This work deals with the application of artificial neural networks to two common problems in spectroscopy: the identification of distorted UV-visible spectra of a specific class of organic compounds, and the quantitative determination of single components in binary mixtures of these compounds. The examined species were six organic indicators, whose spectra are very similar to each other; the trained networks have proven to be very powerful in both applications.

Some structural features of cluster-coordinating cysteines of Clostridium pasteurianum ferredoxin are revealed by 2D TOCSY 1H NMR on the oxidized protein.

Different sets of geminal J coupling constants for the eight beta-CH2 protons in the iron-coordinating cysteines in Clostridium pasteurianum ferredoxin were detected by 2D TOCSY 1H NMR experiments on the oxidized protein. Four resonances were characterized by quite similar high values of J, two more resonances had a J value about half of the former ones, while the last two had extremely low J values. These findings suggest that the cysteines required for cubane symmetry around the iron atoms are constrained into different geometries. The simplified model used for fine tuning of tau m in these TOCSY experiments is also presented and discussed.

Reversible and non-denaturing replacement of iron by cadmium in Clostridium pasteurianum ferredoxin.

Incubation of native, reduced Clostridium pasteurianum ferredoxin with different metals gave a range of modifications in the electronic and EPR spectrum of the protein, or made the signals disappear. The reduced protein, isolated after incubation with different metals under identical conditions (50 microM protein, 1 mM metal, 1 h incubation) was found to contain amounts of foreign metals increasing with their thiophylicity, i.e. Cd2+ >> Zn2+ > Co2+. Little, if any, incorporation was observed for Ni2+, Cu2+, Mn2+ or in the absence of reductant. The activity of substituted ferredoxins in a hydrogenase-coupled assay was proportional to the amount of residual iron, suggesting that the residual iron is present in a population of intact active molecules rather than in partially substituted clusters distributed among individual molecules. The cadmium-substituted ferredoxin did not contain iron, but contained eight cadmium atoms and six labile sulfide atoms/mol. Folding of the isolated, substituted proteins was investigated by CD and 1H-NMR. Both techniques showed retention of the main structural features of the protein upon metal substitution. The rate and extent of the substitution of iron by cadmium were essentially independent of pH, but were found to decrease with increasing ionic strength and to increase with the cadmium concentration. In the cadmium-substituted protein, cadmium was replaced by iron upon incubation with iron and mercaptoethanol in the absence of dithionite. In the presence of dithionite, cadmium was not replaced by iron upon incubation of the cadmium-substituted protein with excess iron and mercaptoethanol. In competition experiments, incubation of iron-containing ferredoxin with stoichiometric amounts of cadmium in the presence of dithionite and excess iron and mercaptoethanol resulted in quantifiable replacement of iron by cadmium. Therefore, substitution of iron by cadmium was only achieved under reducing conditions, and was only reversible in the absence of strong reductants.

Heteronuclear 113Cd-1H NMR study of metal coordination in the human retinoic acid receptor-beta DNA binding domain.

The two zinc fingers of the DNA binding domain of the human retinoic acid receptor-beta were labelled with 113Cd. Two- and three-dimensional heteronuclear nuclear magnetic resonance (NMR) experiments show that the first eight conserved cysteine residues coordinate the two zinc ions tetrahedrally. The ninth conserved cysteine is not involved in metal coordination. In each finger one cysteine exhibits a heteronuclear 113Cd-1H coupling constant substantially smaller than those of the other metal binding cysteines.

Famotidine, the new antiulcero-genic agent, a potent ligand for metal ions.

Potentiometric, polarographic, and spectroscopic results obtained for Cu2+ and Ni(2+)-famotidine systems clearly indicated that this anti-ulcerogenic drug is a very potent chelating agent able to coordinate cupric ion that was at pH below 2. This drug exhibits excellent histamine H2 receptor blocking effects and its effective coordination to metal ions may have significant biological implications. Famotidine is found to be a very effective ligand for Ni2+ ions also.

1H NMR studies on the oxidized ferredoxin from Clostridium pasteurianum.

The 8Fe-8S ferredoxin from Clostridium pasteurianum was investigated by 1D and 2D 1H NMR. Spectra of a well-structured, full native preparation of the oxidized protein in 1 M NaCl at pH 8.0 are presented. Assignments of non-isotropically shifted resonances in the diamagnetic region of the spectrum, namely those of the unique aromatic residues F30 and Y2, are presented for the first time.

The solution structure of a monomeric insulin. A two-dimensional 1H-NMR study of des-(B26-B30)-insulin in combination with distance geometry and restrained molecular dynamics.

The solution conformation of des-(B26-B30)-insulin (DPI) has been investigated by 1H-NMR spectroscopy. A set of 250 approximate interproton distance restraints, derived from two-dimensional nuclear Overhauser enhancement spectra, were used as the basis of a structure determination using distance geometry (DG) and distance-bound driven dynamics (DDD). Sixteen DG structures were optimized using energy minimization (EM) and submitted to short 5-ps restrained molecular dynamics (RMD) simulations. A further refinement of the DDD structure with the lowest distance errors was done by energy minimization, a prolonged RMD simulation in vacuo and a time-averaged RMD simulation. An average structure was obtained from a trajectory generated during 20-ps RMD. The final structure was compared with the des-(B26-B30)-insulin crystal structure refined by molecular dynamics and the 2-Zn crystal structure of porcine insulin. This comparison shows that the overall structure of des-(B26-B30)-insulin is retained in solution with respect to the crystal structures with a high flexibility at the N-terminal part of the A chain and at the N-terminal and C-terminal parts of the B chain. In the RMD run a high mobility of Gly A1, Asn A21 and of the side chain of Phe B25 is noticed. One of the conformations adopted by des-(B26-B30)-insulin in solution is similar to that of molecule 1 (Chinese nomenclature) in the crystal structure of porcine insulin.