Transport kinetics of pseudouridine during hemodialysis and continuous ambulatory peritoneal dialysis.

It has been found that the concentrations of pseudouridine in serum of patients undergoing continuous ambulatory peritoneal dialysis (CAPD) are higher than those in patients undergoing hemodialysis. We analyzed whether this could be caused by a lower rate of transport in CAPD when compared with hemodialysis. Mass transfer area coefficients (MTCs) for urea, creatinine, uric acid, and pseudouridine were determined in nine patients undergoing hemodialysis as dialyzer clearances and in 14 patients undergoing CAPD during a 4-hour dwell with 2 L dialysate with glucose, 70 mmol/L. The theoretical MTC of pseudouridine (TPSI), calculated by extrapolation to its molecular weight by use of the MTC of urea, creatinine, and uric acid, was higher than the observed MTC of pseudouridine, both in hemodialysis (136 vs 112 ml/min, p less than 0.025) and in CAPD (6.9 vs 3.4 ml/min, p less than 0.001). The pseudouridine/TPSI MTC ratio was lower during CAPD than during hemodialysis (0.47 vs 0.83, p less than 0.0005), indicating a lower level of transport during CAPD. In vitro experiments with nuclear magnetic resonance spectroscopy supported the hypothesis of glucose-induced molecular association of pseudouridine. Therefore, dialysate containing 10 mmol/L glucose was compared with that containing 70 mmol/L glucose in eight patients undergoing CAPD. The MTC of pseudouridine was higher during the experiments with dialysate containing 10 mmol/L glucose (3.5 +/- 2.0 ml/min vs 2.7 +/- 1.9 ml/min, p less than 0.05). This was also found for the pseudouridine/TPSI MTC ratio (0.61 vs 0.41, p less than 0.02) and the pseudouridine/creatinine MTC ratio (0.33 vs 0.25, p less than 0.02), favoring glucose-induced decrease of MTC-pseudouridine.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation, identification, and analysis of 4-acetylaminophenol-glucuronide in body fluids of dialyzed renal patients; a molecular mass marker for peritoneal diffusive transport.

A noncharacteristic solute, appearing in gradient elution liquid chromatography (HPLC) profiles of body fluids of dialyzed renal patients, was isolated and identified by preparative HPLC, beta-glucuronidase induced enzymatic peak shift, and mass spectrometry. The compound was shown to be p-acetylaminophenol ('paracetamol')-glucuronide (PG). Serum and peritoneal dialysate PG concentrations were determined in a number of patients. Cuprophan in vivo dialyzer clearances were calculated. Peritoneal membrane mass transfer coefficients (MTC) of PG were calculated and compared with those of molecular mass markers for peritoneal diffusive mass transport studies (urea, creatinine, uric acid, and inulin). By extrapolation of an MTC versus molecular mass calibration line for urea, creatinine, and uric acid it is shown that PG behaves as expected from its molecular mass. We suggest that PG (Mr = 327) is suitable as a molecular mass marker for the molecular mass range between Mr 200 and 500. It may also be used as a marker for diffusive solute transport in hemodialysis treatment. The HPLC gradient elution technique used here appears to be suitable for the simultaneous analysis of the molecular mass markers creatinine, uric acid, and paracetamolglucuronide.

Ultraviolet-absorbing organic anions in uremic serum separated by capillary zone electrophoresis, and quantification of hippuric acid.

Organic anions accumulated in blood serum of patients with chronic renal failure were separated by a novel technique: closed-system capillary zone electrophoresis (CZE) in a pH6 carrier-electrolyte system. Hippuric acid (HA), p-hydroxyhippuric acid, and uric acid were identified by their co-elution with standards prepared in ultrafiltered normal serum and by comparison with the corresponding ultraviolet-detected peaks positively identified in the HPLC analyses. Analysis time for the entire profile is 8 min. Repeatabilities (CVs) of CZE migration times and peak areas of the three acids in serum samples were about 0.7% and 6%, respectively. We quantified HA in 10 ultrafiltered uremic serum samples and compared results with those by a previously described HPLC procedure. The very good agreement further supports the identification of hippuric acid. Accuracy and precision of the CZE method were similar to those for the HPLC gradient-elution method, but analysis time for HA (8 min) is much less than by HPLC (90 min). Our technique is very suitable for selective, rapid analysis for (ultraviolet-absorbing) anionic constituents in ultrafiltered uremic serum, without any sample pretreatment.

Study on the analogy of gel permeation chromatography and cuprophan dialysis for the isolation of uremic 'middle molecules' using model compounds.

Selectivities of substituted nitrobenzenes in gel permeation chromatography and cuprophan membrane dialysis were compared. Glucuronide-, glucoside-, acetic acid- and lactoside-substituted p-nitrobenzenes were chosen as model compounds for so-called 'middle molecules' in uremia. It was found that there was not a single linear relationship between the substituent effects in both processes. This was because of the anomalous behaviour of the model compounds in gel permeation. A combination of adsorption and ionic rejection in the latter technique, for the various solutes, was not encountered in cuprophan membrane dialysis, which appeared to be governed only by the molecular mass or volume of the solute. Therefore, gel permeation seems to be inappropriate for the analysis or isolation for so-called middle molecules in sera of uremic patients. Dialysis or filtration on relatively inert membranes is less susceptible to anomalies.

Biochemical and neurophysiological parameters in hemodialyzed patients with chronic renal failure.

Serum concentrations of accumulated solutes, standard clinical biochemistry, and parameters of clinical neuropathy, were determined in hemodialyzed patients with chronic renal failure. Analyses by high-performance liquid chromatography included creatinine, pseudouridine, urate, p-hydroxyhippuric acid, hippuric acid, indoxylsulfate, tryptophan, tyrosine, 3-indoleacetic acid, and a number of as-yet unidentified solutes. Standard biochemical parameters were measured; aluminium, parathyroid hormone, serum electrolytes and enzymes, hemoglobin, bilirubin, phosphate and urea. Measures of clinical neuropathy were: maximal motor nerve conduction velocities, and Hoffmann reflex latency. Several solutes had higher concentrations when nerve function was impaired. Serum total LDH, and total calcium levels correlated positively with values of the Hoffmann reflex, as did serum hippuric acid concentrations. Concentrations of p-hydroxyhippuric acid and two fluorescent compounds correlated negatively with motor nerve conduction velocities. In principal component analysis a number of 'organic acid-like' substances, like hippuric acid and p-hydroxyhippuric acid, were shown to associate multivariately with the neurophysiological variables while urea, creatinine, urate and phosphate were not.

Effect of hemodialysis on serum concentrations of HPLC-analyzed accumulating solutes in uremia.

The concentration changes, during hemodialysis treatment, of 18 characteristic uremia compounds, analyzed by high-pressure liquid chromatography in sera of renal patients were studied. Pre- to postdialysis concentration ratios (dialysis ratio, D) varied from 0.83 to 3.04 for the different solutes. A division into three solute groups, on the basis of their D values, could be rationalized qualitatively from data on protein binding and dialyzer clearance. One group showed low dialysis ratios which could be explained from plasma protein binding. The second group had intermediate D values, comparable to those of creatinine. For some of the members of the third group, high D values might indicate a compartmentalization and resistance to mass transfer across biological membranes. Among the latter are the tubularly secreted hippuric acid and p-hydroxyhippuric acid. For most of the (protein-bound) solutes, protein binding was shown to decrease during hemodialysis. Protein binding levels were higher after dialysis only for hippuric acid and the as yet unidentified fluorescent solute designated UFK8. In conclusion, the change of serum concentrations and of protein binding resulting from hemodialysis treatment are presented and are compared for 18 accumulating solutes in sera of patients with end-stage renal failure.

Are the classical markers sufficient to describe uremic solute accumulation in dialyzed patients? Hippurates reconsidered.

Interdependencies of accumulated solutes, analyzed by liquid chromatography in dialyzed and non-dialyzed patients, were studied by multivariate statistical analysis. In principal component analysis, three principal components (PC1-PC3) were retained from the data on 22 accumulated compounds in dialyzed patients, whereas only one principal component was retained from analogous data of a non-dialyzed patient group. PC1 in the dialyzed patient group comprises concentrations of hippuric acid, p-hydroxyhippuric acid, tryptophan, and five unidentified fluorescent solutes in serum. Concentrations of the classical markers urea, uric acid, creatinine, and phosphate were closely related to PC2 in these patients. Indoleacetic acid and two unidentified fluorescent compounds constitute PC3. The compounds associated with the groups found by principal component analysis may be characterized by chemical structure and by the mechanism of their excretion via the remaining nephrons of dialyzed patients. In the non-dialyzed group, most of the solutes could be described by a single PC. This PC and PC1 from the dialyzed group correlated significantly with residual renal function, and with total ultraviolet absorbance and total fluorescence emission. The data suggest that it is of value to introduce a marker of uremic solute retention in addition to urea, to account for renal-function-related "organic-acid-like" compounds that are excreted by renal tubular secretion in dialyzed patients. The hippurates may serve this purpose.

Hexachlorobenzene and 1,1-di(4-chlorophenyl)-2,2-dichloroethene in serum of uremic patients and healthy persons: determination by capillary gas chromatography and electron capture detection.

The concentrations of organochlorine pesticides in serum have been determined by GC and electron capture detection. Studies were performed in 7 nondialyzed, 10 dialyzed and 6 healthy persons. Only hexachlorobenzene (HCB) and 1,1-di(4-chlorophenyl)-2,2-dichloroethene (p,p'-DDE) were consistently present. No interference by other pesticides or polychlorinated biphenyls was found by checking with GC-MS. For the nondialyzed uremic patients the average HCB concentration was 16.2 nmol/l (sigma = 6.7, n = 7), and the p,p'-DDE level 26.4 nmol/l (sigma = 31.4, n = 7). For the dialyzed uremic patients the average HCB level was 15.5 nmol/l (sigma = 11.2, n = 10) before dialysis and 17.2 nmol/l (sigma = 14.4, n = 8) after dialysis, the concentration of p,p'-DDE was 27.0 nmol/l (sigma = 38.4, n = 10) before dialysis and 28.0 nmol/l (sigma = 37.4, n = 8) after dialysis. For the healthy persons the average concentration of HCB was 7.7 nmol/l (sigma = 1.8, n = 6) and the concentration of p,p'-DDE was 20.1 nmol/l (sigma = 10.4, n = 6). HCB concentrations were significantly higher in serum of dialyzed and nondialyzed uremic patients than in controls (Wilcoxon's test).

Liquid-chromatographic profiling of solutes in serum of uremic patients undergoing hemodialysis and chronic ambulatory peritoneal dialysis (CAPD); high concentrations of pseudouridine in CAPD patients.

Using "high performance" liquid chromatography, we studied non-protein-bound fractions and total concentrations of 18 solutes accumulating in sera from a group of 12 patients who were undergoing chronic ambulatory peritoneal dialysis (CAPD) and in predialysis sera from a group of 15 hemodialysis (HD) patients. We monitored longitudinal changes in solute concentrations for two patients with respect to change of therapy between HD and CAPD. The concentrations of pseudouridine (P less than 0.001), uric acid (P less than 0.001), and an unknown fluorescent solute, "UKF3" (P less than 0.01), differed in sera of HD and CAPD patients. When standardized with respect to serum creatinine concentrations, the concentration of the transfer-RNA catabolite, pseudouridine, was significantly (P less than 0.0001) higher in sera of CAPD patients than in HD patients, suggesting an increase in turnover of transfer RNA. In stepwise linear discriminant analysis, the combination of pseudouridine and the probably biochemically related fluorescent unknown, UKF3, contributed most to the differentiation between sera from CAPD and HD patients.

Screening of UV-absorbing solutes in uremic serum by reversed phase HPLC--change of blood levels in different therapies.

In order to screen UV-absorbing solutes in large numbers of uremic serum samples, an automated liquid chromatographic method was developed. The method proved to be reliable and reproducible in more than 500 analyses. HPLC separation was performed using gradient elution on a 25-cm Ultrasphere Octyl reversed phase column, with 5 microns particles. Characteristic profiles for the uremic state were obtained in the analyses of serum samples of 43 uremic patients before and just after artificial kidney treatment; hemodialysis (n = 14), hemodiafiltration (n = 13) and hemofiltration (n = 16). In these profiles 20-40 peaks were resolved of which nine were 'quantitated' by peak height relative to a standard. Of these solutes creatinine, uracil, uric acid, hypoxanthine, indoxylsulfate, tryptophan and hippuric acid were identified. The heterogeneity of the population of uremic patients, with respect to the UV-absorbing solutes, was estimated. Significant differences of solute blood level changes during hemodialysis, hemodiafiltration and hemofiltration, were observed.

Characterization of uremic "middle molecular" fractions by gas chromatography, mass spectrometry, isotachophoresis, and liquid chromatography.

Uremic ultrafiltrates (and normal serum, for comparison) were fractionated by means of gel filtration. The collected fractions were further investigated by combined analytical techniques: "high-performance" liquid chromatography, gas chromatography, mass spectrometry, and isotachophoresis. Ultrafiltrate fractions in the so-called middle molecular mass region (Mr 500-2000) contained a considerable amount of substances of low molecular mass, such as carbohydrates, organic acids, amino acids, and ultraviolet absorbing solutes. Ultraviolet absorbance in the "middle molecular mass region" of the gel chromatogram is mainly due to the presence of these rather low-molecular-mass solutes. Therefore this signal is not a quantitative measure of molecules with a "middle" molecular mass.

Chemical ionization mass spectrometry of trimethylsilylated carbohydrates and organic acids retained in uremic serum.

After appropriate sample pretreatment and derivatization, uremic serum was investigated by combined high resolution gas chromatography and mass spectrometry, using both electron impact and chemical ionization methods. Electron impact and chemical ionization spectra of a number of identified (trimethylsilylated) carbohydrates and organic acids are compared. The utilization of chemical ionization mass spectrometry, with isobutane as the reagent gas, is discussed in detail. The influence of the reagent gas pressure on the total ion current and on the spectral appearance was studied. The identification of compounds, based on electron impact mass spectral data, was confirmed and often aided appreciably by using this technique. The chemical ionization spectra of trimethylsilyated alditols and aldonic acids, as well as of other organic acids showed protonated molecular ions, whereas aldoses did not. Differences with electron impact spectra are found mainly in the high mass region. The loss of one or more trimethylsilanol groups becomes the predominating fragmentation route at higher reagent gas pressures.

Profiling of uremic serum by high-resolution gas chromatography-electron-impact, chemical ionization mass spectrometry.

A fast and reliable procedure for gas chromatographic profiling of components in ultrafiltrated uremic serum has been developed, using glass capillary columns. Sample pretreatment consists of ultrafiltration, evaporation and silylation. Some twenty components are identified by electron-impact and chemical ionization mass spectrometry. A comparison is made between profiles of sera from a series of uremic patients, before and after hemodialysis, and from non-uremic sera. Significant differences are found between these profiles. A "dialysis ratio" is introduced as a parameter for the removal of retained components by hemodialysis treatment.