Optical Real-Time Cardiorenal Toxin Uric Acid Measurement During Hemodialysis Using a Miniaturized Optical Sensor.

Patients with chronic kidney disease (CKD) are at higher cardiovascular risk than the general population. Cardiovascular diseases, vascular calcification among them, are the leading cause of death in these patients. Factors influencing vascular calcification are oxidative stress, inflammation, and accumulation of uremic toxins during CKD. Uric acid is a cardiorenal toxin that accumulates in the case of kidney malfunction. The primary therapy for replacing kidney function and removing toxins from end-stage renal disease patients is hemodialysis. Effective removal of toxins can be estimated by blood or dialysate lab analysis or optical monitoring. In this study, the authors tested a miniaturized optical sensor for monitoring uric acid levels and removal for the first time in a more extensive clinical study, including Hemodialysis (HD) and Post-dilutional online hemodiafiltration (HDF) procedures with different settings in Tallinn, Estonia. The results (Mean±SD, Lab vs. Sensor) of the uric acid concentration 57.20±34.05 vs. 57.22±33.09 µmol/L, reduction ratio 68.72±10.91 vs. 67.89±12.48 %, and total removed amount 7.00±2.10 vs. 7.33±2.29 mmol did not differ significantly from the values obtained from the clinical laboratory (p<0.05).Clinical Relevance-During this study, a miniaturized optical sensor was tested for the first time in the clinic in different dialysis settings. The results confirm that the sensor is reliable for regularly monitoring cardiorenal toxin uric acid removal during hemodialysis.

Treatment with Paracetamol Can Interfere with the Intradialytic Optical Estimation in Spent Dialysate of Uric Acid but Not of Indoxyl Sulfate.

Optical online methods are used to monitor the haemodialysis treatment efficiency of end stage kidney disease (ESKD) patients. The aim of this study was to analyse the effect of the administration of UV-absorbing drugs, such as paracetamol (Par), on the accuracy of optical monitoring the removal of uremic toxins uric acid (UA) and indoxyl sulfate (IS) during standard haemodialysis (HD) and haemodiafiltration (HDF) treatments. Nine patients received Par in daily dosages 1−4 g for 30 sessions. For 137 sessions, in 36 patients the total daily dosage of UV-absorbing drugs was less than 500 mg, and for 6 sessions 3 patients received additional UV-absorbing drugs. Par administration slightly affected the accuracy of optically assessed removal of UA expressed as bias between optically and laboratory-assessed reduction ratios (RR) during HD but not HDF employing UV absorbance of spent dialysate (p < 0.05) at 295 nm wavelength with the strongest correlation between the concentration of UA and absorbance. Corresponding removal of IS based on fluorescence at Ex280/Em400 nm during HD and HDF was not affected. Administration of UV-absorbing drugs may in some settings influence the accuracy of optical assessments in spent dialysate of the removal of uremic solutes during haemodialysis treatment of ESKD patients.

Optical Method and Biochemical Source for the Assessment of the Middle-Molecule Uremic Toxin ß2-Microglobulin in Spent Dialysate.

Optical monitoring of spent dialysate has been used to estimate the removal of water-soluble low molecular weight as well as protein-bound uremic toxins from the blood of end stage kidney disease (ESKD) patients. The aim of this work was to develop an optical method to estimate the removal of ß2-microglobulin (ß2M), a marker of middle molecule (MM) uremic toxins, during hemodialysis (HD) treatment. Ultraviolet (UV) and fluorescence spectra of dialysate samples were recorded from 88 dialysis sessions of 22 ESKD patients, receiving four different settings of dialysis treatments. Stepwise regression was used to obtain the best model for the assessment of ß2M concentration in the spent dialysate. The correlation coefficient 0.958 and an accuracy of 0.000 ± 0.304 mg/L was achieved between laboratory and optically estimated ß2M concentrations in spent dialysate for the entire cohort. Optically and laboratory estimated reduction ratio (RR) and total removed solute (TRS) of ß2M were not statistically different (p > 0.35). Dialytic elimination of MM uremic toxin ß2M can be followed optically during dialysis treatment of ESKD patients. The main contributors to the optical signal of the MM fraction in the spent dialysate were provisionally identified as tryptophan (Trp) in small peptides and proteins, and advanced glycation end-products.

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).

Free Pentosidine Assessment Based on Fluorescence Measurements in Spent Dialysate.

The aim of this study was to primarily explore the relationship between free pentosidine and the fluorescence properties of spent dialysate, and also to develop a model to assess the levels of free pentosidine in spent dialysate based on the fluorescence measurements. First, 40 patients (20 females and 20 males) were examined during 40 dialysis sessions. High-pressure liquid chromatography (HPLC) was used to measure the free pentosidine concentrations from the spent dialysate. The full fluorescence spectra of the spent dialysates were recorded and single- and multi-wavelength (MW) models were developed. The average free pentosidine concentrations in the spent dialysate measured by HPLC at the start and end of the dialysis session were (mean ± SD) 4.25 ± 3.11 and 0.94 ± 0.69 µg/L respectively. The removal ratios (RRs) between RR_lab and RR_MW were statistically similar (p > 0.2). The concentration of free pentosidine and the RR can therefore be estimated from the spent dialysate when utilising fluorescence measurements.

4-Pyridoxic Acid in the Spent Dialysate: Contribution to Fluorescence and Optical Monitoring.

AIM: In this work we estimated the contribution of the fluorescence of 4-pyridoxic acid (4-PA) to the total fluorescence of spent dialysate with the aim of evaluating the on-line monitoring of removal of this vitamin B-6 metabolite from the blood of patients with end-stage renal disease (ESRD). METHODS: Spectrofluorometric analysis of spent dialysate, collected from hemodialysis and hemodiafiltration sessions of 10 patients receiving regularly pyridoxine injections after dialysis treatment, was performed in the range of Ex/Em 220-500 nm. 4-PA in dialysate samples was identified and quantified using HPLC with fluorescent and MS/MS detection. RESULTS: Averaged HPLC chromatogram of spent dialysate had many peaks in the wavelength region of Ex320/Em430 nm where 4-PA was the highest peak with contribution of 42.2±17.0% at the beginning and 47.7±18.0% in the end of the dialysis. High correlation (R = 0.88-0.95) between 4-PA concentration and fluorescence intensity of spent dialysate was found in the region of Ex310-330/Em415-500 nm, respectively. CONCLUSION: 4-PA elimination from the blood of ESRD patients can be potentially followed using monitoring of the fluorescence of the spent dialysate during dialysis treatments.

Is Fluorescence Valid to Monitor Removal of Protein Bound Uremic Solutes in Dialysis?

The aim of this study was to evaluate the contribution and removal dynamics of the main fluorophores during dialysis by analyzing the spent dialysate samples to prove the hypothesis whether the fluorescence of spent dialysate can be utilized for monitoring removal of any of the protein bound uremic solute. A high performance liquid chromatography system was used to separate and quantify fluorophoric solutes in the spent dialysate sampled at the start and the end of 99 dialysis sessions, including 57 hemodialysis and 42 hemodiafiltration treatments. Fluorescence was acquired at excitation 280 nm and emission 360 nm. The main fluorophores found in samples were identified as indole derivatives: tryptophan, indoxyl glucuronide, indoxyl sulfate, 5-hydroxy-indoleacetic acid, indoleacetyl glutamine, and indoleacetic acid. The highest contribution (35 ± 11%) was found to arise from indoxyl sulfate. Strong correlation between contribution values at the start and end of dialysis (R2 = 0.90) indicated to the stable contribution during the course of the dialysis. The reduction ratio of indoxyl sulfate was very close to the decrease of the total fluorescence signal of the spent dialysate (49 ± 14% vs 51 ± 13% respectively, P = 0.30, N = 99) and there was strong correlation between these reduction ratio values (R2 = 0.86). On-line fluorescence measurements were carried out to illustrate the technological possibility for real-time dialysis fluorescence monitoring reflecting the removal of the main fluorophores from blood into spent dialysate. In summary, since a predominant part of the fluorescence signal at excitation 280 nm and emission 360 nm in the spent dialysate originates from protein bound derivatives of indoles, metabolites of tryptophan and indole, the fluorescence signal at this wavelength region has high potential to be utilized for monitoring the removal of slowly dialyzed uremic toxin indoxyl sulfate.

Estimation of removed uremic toxin indoxyl sulphate during hemodialysis by using optical data of the spent dialysate.

The aim of this study was to explore the possibility to determine the amount of total removed Indoxyl Sulphate (TR_IS) during dialysis session, an optical method utilizing absorbance and fluorescence spectral data of the spent dialysate was used. Eight uremic patients from Linköping, Sweden and 10 from Tallinn, Estonia, were studied during dialysis treatments. Dialysate samples were taken during each treatment and analyzed at a laboratory. Fluorescence and absorbance spectra of the spent dialysate were measured with spectrofluorophotometer and spectrophotometer. The spectral values were transformed into IS concentration using multiple linear regression model from the total material noted as optical method (Opt). IS concentration was estimated using high-performance liquid chromatography (HPLC) method as a reference. TR_IS values were calculated. Achieved results were compared regarding mean values and SD and collated with the amount of total removed urea value (TR_Urea) for the same dialysis procedures. Mean TR value ± SD (mg) for urea was 28 947 ± 9 241; TR for IS was 151.4 ± 87.3 estimated by HPLC and 149.4 ± 84.9 estimated by Opt. The TR_IS values were not significantly different (p ≤ 0.05). This study indicates, that it is possible to estimate TR_IS using only spectral values of the spent dialysate and the parameter can be used for quantifying the elimination of protein bound uremic toxins during the dialysis procedure.

The transcriptional repressor domain of Gli3 is intrinsically disordered.

The transcription factor Gli3 is acting mainly as a transcriptional repressor in the Sonic hedgehog signal transduction pathway. Gli3 contains a repressor domain in its N-terminus from residue G106 to E236. In this study we have characterized the intracellular structure of the Gli3 repressor domain using a combined bioinformatics and experimental approach. According to our findings the Gli3 repressor domain while being intrinsically disordered contains predicted anchor sites for partner interactions. The obvious interaction partners to test were Ski and DNA; however, with both of these the structure of Gli3 repressor domain remained disordered. To locate residues important for the repressor function we mutated several residues within the Gli3 repressor domain. Two of these, H141A and H157N, targeting predicted helical regions, significantly decreased transcriptional repression and thus identify important functional parts of the domain.

Paracetamol interference in uric Acid levels in uremic patients revealed by monitoring spent dialysate.

The aim of this study was to assess removal dynamics of paracetamol (PAR), as an extraordinary chromophore in spent dialysate, upon the optical monitoring of dialysis of end-stage renal disease patients with inflammation complications. Seven dialysis sessions of different patients were followed to whom PAR was used as a pain reliever or antipyretic. Spent dialysate was sampled hourly and analyzed using HPLC with MS/MS and UV detection. Quantitative calculations were made on the basis of the peak areas on the chromatograms at 280 nm for uric acid (UA) and 254 nm for PAR and its metabolites (PAR-M). Peaks of UA, PAR, PAR-glucuronide, and PAR-sulphate were identified on the basis of specific mass spectra. Removal of PAR was found to be proportional to that of uric acid if intake of the drug by patient occurred half a day before dialysis. But disturbances of the UV-absorbance curves at 280 nm were observed related to rise of UA concentration in spent dialysate when PAR was taken by patients in the course of dialysis. The mechanism of such relation remains unknown. It was concluded that possible benefits and risks of treatment of uremic patients with paracetamol-containing drugs may need to be reassessed.

Do only small uremic toxins, chromophores, contribute to the online dialysis dose monitoring by UV absorbance?

The aim of this work was to evaluate the contributions of the main chromophores to the total UV absorbance of the spent dialysate and to assess removal dynamics of these solutes during optical on-line dialysis dose monitoring. High performance chromatography was used to separate and quantify UV-absorbing solutes in the spent dialysate sampled at the start and at the end of dialysis sessions. Chromatograms were monitored at 210, 254 and 280 nm routinely and full absorption spectra were registered between 200 and 400 nm. Nearly 95% of UV absorbance originates from solutes with high removal ratio, such as uric acid. The contributions of different solute groups vary at different wavelengths and there are dynamical changes in contributions during the single dialysis session. However, large standard deviation of the average contribution values within a series of sessions indicates remarkable differences between individual treatments. A noteworthy contribution of Paracetamol and its metabolites to the total UV absorbance was determined at all three wavelengths. Contribution of slowly dialyzed uremic solutes, such as indoxyl sulfate, was negligible.

Concentration of uric acid removed during dialysis. Estimated by multi wavelength and processed ultra violet absorbance spectra.

The aim of this study was to estimate the concentration of uric acid (UA) optically by using original and processed ultra violet (UV) absorbance spectra's of the spent dialysate. Also the effect of using several wavelengths for estimation was examined.

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.

A multicentre study of an enhanced optical method for measuring concentration of uric acid removed during dialysis.

The aim of this study was to compare concentration measurements of uric acid (UA) removed during dialysis by two algorithms based on UV-absorbance and the 1st derivate of UV absorbance. Ten uremic patients from Tallinn and ten from Linköping, during 30+40 haemodialysis treatments, were followed at the Departments of Dialysis and Nephrology at North-Estonian Medical Centre and at Linköping University Hospital. The dialysate samples were taken and analyzed by means of UA concentration at the chemical laboratory and with a double-beam spectrophotometer. UV absorbance and derivate of UV absorbance was transformed into UA concentration in the spent dialysate using the regression models from the calibration set of material, noted as UV-absorbance (UV_A) and the 1st derivate of UV absorbance (UV_D) method. These models were tested on validation set of material and concentrations of UA from the two methods were compared regarding mean values and SD. Mean concentration of UA were 52.7 +/- 25.0 micromol/l measured at the chemical laboratory (UA_Lab), 54.9 +/- 23.8 micromol/l determined by UV_A and 52.9 +/- 23.0 micromol/l determined by UV_D. The results of mean concentrations were not significantly different (p > or = 0.54). The systematic errors were -7.8 % and -3.3% and random errors were 15.8 % and 10.4 % using UV_A and UV_D respectively. The systematic and random errors were significantly different (p < 0.05) indicating that the new algorithm enables more accurate UA estimation.

Towards the electrospray ionization mass spectrometry ionization efficiency scale of organic compounds.

An approach that allows setting up under predefined ionization conditions a rugged self-consistent quantitative experimental scale of electrospray ionization (ESI) efficiencies of organic compounds is presented. By ESI ionization efficiency (IE) we mean the efficiency of generating gas-phase ions from analyte molecules or ions in the ESI source. The approach is based on measurement of relative ionization efficiency (RIE) of two compounds (B1 and B2) by infusing a solution containing both compounds at known concentrations (C1 and C2) and measuring the mass-spectrometric responses of the protonated forms of the compounds (R1 and R2). The RIE of B1 and B2 is expressed as logRIE(B1, B2) = log[(R1 . C2)/(C1 . R2)]. The relative way of measurement leads to cancellation of many of the factors affecting IE (ESI source design, voltages in the source and ion transport system, solvent composition, flow rates and temperatures of the nebulizing and drying gases). Using this approach an ESI IE scale containing ten compounds (esters and aromatic amines) and spanning over 4 logRIE units has been compiled. The consistency of the scale (the consistency standard deviation of the scale is s = 0.16 logRIE units) was assured by making measurements using different concentration ratios (at least 6-fold concentration ratio range) of the compounds and by making circular validation measurements (the logRIE of any two compounds was checked by measuring both against a third compound).

Optical dialysis adequacy sensor: wavelength dependence of the ultra violet absorbance in the spent dialysate to the removed solutes.

A need for dialysate-based, on-line, continuous monitoring systems for the control of dialysis efficiency and the prevention of dialysis-associated complications is arisen due to increasing number of dialysis patients and related treatment quality requirements. The aim of this study was to investigate the wavelength dependence between the the ultra-violet (UV) absorbance in the spent dialysate and the retained solutes removed during the hemodialysis in order to explain possibilities to estimate removal of the solutes by the optical dialysis adequacy sensor. Ten uremic patients, during 30 hemodialysis treatments, were followed at the Department of Dialysis and Nephrology, North-Estonian Regional Hospital. The dialysate samples were taken and analyzed with spectrophotometer to get absorbance spectra. The results confirm previous studies considering similarity for the UV-spectrum on the spent dialysate samples during a single dialysis session indicating presence of the same type of chromophores in the spent dialysate removed from the patient's blood for different patients groups. At the same time the highest correlation in the spent dialysate for urea, creatinine, potassium, and phosphate was obtained at the wavelength 237 nm that is a new finding compared to earlier results. The highest correlation between the UV-absorbance and uric acid in the spent dialysate was obtained at the wavelength 294 nm. Presence of at least two different wavelength ranges may add selectivity for monitoring several compounds. Our study indicates that the technique has a potential to estimate the removal of retained substances.

Maximum entropy reconstruction of joint phi, psi-distribution with a coil-library prior: the backbone conformation of the peptide hormone motilin in aqueous solution from phi and psi-dependent J-couplings.

In this paper, we present a new method for structure determination of flexible "random-coil" peptides. A numerical method is described, where the experimentally measured 3J(H(alpha)Nalpha) and [3J(H(alpha)Nalpha+1 couplings, which depend on the phi and psi dihedral angles, are analyzed jointly with the information from a coil-library through a maximum entropy approach. The coil-library is the distribution of dihedral angles found outside the elements of the secondary structure in the high-resolution protein structures. The method results in residue specific joint phi,psi-distribution functions, which are in agreement with the experimental J-couplings and minimally committal to the information in the coil-library. The 22-residue human peptide hormone motilin, uniformly 15N-labeled was studied. The 3J(H(alpha)-N(i+1)) were measured from the E.COSY pattern in the sequential NOESY cross-peaks. By employing homodecoupling and an in-phase/anti-phase filter, sharp H(alpha)-resonances (about 5 Hz) were obtained enabling accurate determination of the coupling with minimal spectral overlap. Clear trends in the resulting phi,psi-distribution functions along the sequence are observed, with a nascent helical structure in the central part of the peptide and more extended conformations of the receptor binding N-terminus as the most prominent characteristics. From the phi,psi-distribution functions, the contribution from each residue to the thermodynamic entropy, i.e., the segmental entropies, are calculated and compared to segmental entropies estimated from 15N-relaxation data. Remarkable agreement between the relaxation and J-couplings based methods is found. Residues belonging to the nascent helix and the C-terminus show segmental entropies, of approximately -20 J K(-1) mol(-1) and -12 J K(-1) mol(-1), respectively, in both series. The agreement between the two estimates of the segmental entropy, the agreement with the observed J-couplings, the agreement with the CD experiments, and the assignment of population to sterically allowed conformations show that the phi,psi-distribution functions are indeed meaningful and useful descriptions of the conformational preferences for each residue in this flexible peptide.

Optical dialysis adequacy sensor: contribution of chromophores to the ultra violet absorbance in the spent dialysate.

Several on-line methods have been developed to standardize the assessment of dialysis adequacy. Earlier studies have demonstrated that on-line monitoring of total ultra violet (UV) absorbance in spent dialysate can be utilized to follow continuously a single hemodialysis session. The aim of this study was to investigate the contribution of different compounds, acting as chromophores, to the UV-absorbance in the spent dialysate in order to explain origin of the cumulative and integrated UV-absorbance measured by the optical dialysis adequacy sensor. Four uremic patients, during 12 hemodialysis treatments, were followed by the optical dialysis adequacy sensor using the wavelength of 280 mn. The dialysate samples were taken and analyzed using reversed phase high performance liquid chromatography (HPLC). The total number of detected peaks from the HPLC gradient separation profiles measured at the wavelength 280 nm for the samples collected 10 mm after the start of hemodialysis (Mean +/- SD) was 38 +/- 6. The relative contribution from the area of 10 main peaks to the total area of all detected peaks in percentage was 91.01 +/- 2.52 %. The optical dialysis adequacy sensor provides continuous, on-line hemodialysis measurements and may immediately identify and alert to any deviations in the dialysis. Our study indicates that there exists a number of prevalent compounds that are the main cause of the cumulative and integrated UV- absorbance.