Factors affecting counteraction by methylamines of urea effects on aldose reductase.
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The concentration of urea in renal medullary cells is high enough to affect enzymes seriously by reducing Vmax or raising Km, yet the cells survive and function. The usual explanation is that the methylamines found in the renal medulla, namely glycerophosphocholine and betaine, have actions opposite to those of urea and thus counteract its effects. However, urea and methylamines have the similar (not counteracting) effects of reducing both the Km and Vmax of aldose reductase (EC 1.1.1.21), an enzyme whose function is important in renal medullas. Therefore, we examined factors that might determine whether counteraction occurs, namely different combinations of assay conditions (pH and salt concentration), methylamines (glycerophosphocholine, betaine, and trimethylamine N-oxide), substrates (DL-glyceraldehyde and D-xylose), and a mutation in recombinant aldose reductase protein (C298A). We find that Vmax of both wild-type and C298A mutant generally is reduced by urea and/or the methylamines. However, the effects on Km are much more complex, varying widely with the combination of conditions. At one extreme, we find a reduction of Km of wild-type enzyme by urea and/or methylamines that is partially additive, whereas at the other extreme we find that urea raises Km for D-xylose of the C298A mutant, betaine lowers the Km, and the two counteract in a classical fashion so that at a 2:1 molar ratio of betaine to urea there is no net effect. We conclude that counteraction of urea effects on enzymes by methylamines can depend on ion concentration, pH, the specific methylamine and substrate, and identity of even a single amino acid in the enzyme. (+info)
Change from conventional haemodiafiltration to on-line haemodiafiltration.
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BACKGROUND: On-line haemodiafiltration (HDF) is a technique which combines diffusion with elevated convection and uses pyrogen-free dialysate as a replacement fluid. The purpose of this study was to evaluate the difference between conventional HDF (1-3 l/h) and on-line HDF (6-12 l/h). METHODS: The study included 37 patients, 25 males and 12 females. The mean age was 56.5 +/- 13 years and duration of dialysis was 62.7 +/- 49 months. Three patients dropped out for transplantation, three patients died and three failed to complete the study period. Initially all patients were on conventional HDF with high-flux membranes over the preceding 34 +/- 32 months. Treatment was performed with blood flow (QB) 402 +/- 41 ml/min, dialysis time (Td) 187 min, dialysate flow (QD) 654 +/- 126 ml/min and replacement fluid (Qi) 4.0 +/- 2 l/session. Patients were changed to on-line HDF with the same filtre and dialysis time, QD 679 +/- 38 ml/min (NS), QB 434 +/- 68 ml/min (P < 0.05) and post-dilutional replacement fluid 22.5 +/- 4.3 l/session (P < 0.001). We compared conventional HDF with on-line HDF over a period of 1 year. Dialysis adequacy was monitored according to standard clinical and biochemical criteria. Kinetic analysis of urea and beta2-micro-globulin (beta2m) was performed monthly. RESULTS: Tolerance was excellent and no pyrogenic reactions were observed. Pre-dialysis sodium increased 2 mEq/l during on-line HDF. Plasma potassium, pre- and post-dialysis bicarbonate, uric acid, phosphate, calcium, iPTH, albumin, total proteins, cholesterol and triglycerides remained stable. The mean plasma beta2m reduction ratio increased from 56.1 +/- 8.7% in conventional HDF to 71.1 +/- 9.1% in on-line HDF (P < 0.001). The pre-dialysis plasma beta2m decreased from 27.4 +/- 8.1 to 24.2 +/- 6.5 mg/l (P < 0.01). Mean Kt/V (Daugirdas 2nd generation) was 1.35 +/- 0.21 in conventional HDF compared with 1.56 +/- 0.29 in on-line HDF (P < 0.01), Kt/Vr (Kt/V taking into consideration post-dialysis urea rebound) 1.12 +/- 0.17 vs 1.26 +/- 0.20 (P < 0.01), BUN time average concentration (TAC) 44.4 +/- 9 vs 40.6 +/- 10 mg/dl (P < 0.05) and protein catabolic rate (PCR) 1.13 +/- 0.22 vs 1.13 +/- 0.24 g/kg (NS). There was a significant increase in haemoglobin (10.66 +/- 1.1 vs 11.4 +/- 1.5) and haematocrit (32.2 +/- 2.9 vs 34.0 +/- 4.4%), P < 0.05, during the on-line HDF period, which allowed a decrease in the erythropoietin doses (3861 +/- 2446 vs 3232 +/- 2492 UI/week), (P < 0.05). Better blood pressure control (MAP 103.8 +/- 15 vs 97.8 +/- 11 mmHg, P < 0.01) and a lower percentage of patients requiring antihypertensive drugs were also observed. CONCLUSION: The change from conventional HDF to on-line HDF results in increased convective removal and fluid replacement (18 l/session). During on-line HDF treatment, dialysis dose was increased for both small and large molecules with a decrease in uraemic toxicity level (TAC). On-line HDF provided a better correction of anaemia with lower dosages of erythropoietin. Finally, blood pressure was easily controlled. (+info)
A chemically-defined medium for the growth of a ureolytic strain of Streptococcus faecium.
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A chemically-defined medium was developed which supported growth of Streptococcus faecium and permitted synthesis of urease. This streptococcus cannot utilize ammonia and needs a complex medium, but its requirements are probably provided in the rumen. The specific activity of urease was inversely related to growth and in no medium was there high growth and high urease activity. Anaerobic culture and the presence of urea in the medium were essential for urease activity, but not for growth. (+info)
Metabolic capacity for L-citrulline synthesis from ammonia in rat isolated colonocytes.
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Ammonia is present at high concentration in the colon lumen and is considered a colon cancer suspect. Furthermore, ammonia usually eliminated by the liver in the ornithine cycle is considered highly toxic to cerebral function when present in excess in the blood plasma. Therefore, the metabolic pathways involved in ammonia metabolism in colonocytes were studied in the present study. Rat colonocytes were found equipped with low carbamoylphosphate synthase I activity, high ornithine carbamoyltransferase and arginase activities and low argininosuccinate synthase activity. High (10 and 50 mmol/l) NH4Cl concentrations but not low concentrations (1 and 5 mmol/l) were found able to increase respectively 3- and 10-fold the conversion of radioactive L-arginine to L-citrulline. In contrast, very low capacity for L-citrulline conversion to L-arginine is found in colonocytes. It is concluded that an incomplete ornithine cycle is operative in colonocytes which results in ammonia stimulated L-citrulline production. The contribution of this metabolic pathway in relation to ammonia detoxication by colonocytes is discussed. (+info)
Peritoneal dialysis adequacy: a model to assess feasibility with various modalities.
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BACKGROUND: The current standard of adequacy for peritoneal dialysis (PD) is to provide a weekly normalized urea clearance (Kt/V) of 2.0 or more and a creatinine clearance (CCr) of 60 liter/1.73 m2 or more. As native renal function is lost, it is important to determine the effectiveness of the available therapeutic modalities in achieving these goals. METHODS: A model to assess our ability to provide a weekly Kt/Vurea of 2.0 or more and a CCr of 60 liter/1.73 m2 or more to anuric patients undergoing continuous ambulatory PD (CAPD) and automated PD (PD Plus) was developed. The body surface area (BSA) distribution was obtained from 38,768 patients undergoing dialysis during January 1997. The distribution of peritoneal transport rates (PTRs) was obtained from 2531 peritoneal equilibration tests performed during 1996. The weekly Kpt/Vurea was calculated for the various PTR groups and the range of BSA with four PD prescriptions: CAPD 8 liters, CAPD 10 liters, PD Plus 12 liters, and PD Plus 15 liters, using a previously validated kinetic program (PackPD). RESULTS: The predicted percentage of patients capable of achieving the adequacy goals for Kt/V and CCr, respectively, were 24.8 and 11. 2 for CAPD 8 liters, 54.2 and 33.0 for CAPD 10 liters, 77.8 and 54.9 for PD Plus 12 liters, and 93.2 and 72.9 for PD Plus 15 liters. CONCLUSIONS: Most patients can attain the current adequacy standards of therapy with automated PD, but few (less than 25%) can do so with standard CAPD in the absence of residual renal function. (+info)
Gender differences in normalized clearances in CAPD: role of body size and normalizing parameters.
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OBJECTIVE: To compare raw (not normalized) and normalized urea and creatinine clearances between women and men on continuous ambulatory peritoneal dialysis (CAPD). To study whether potential gender differences are due to the normalization process. DESIGN: Retrospective analysis of clearance studies. SETTING: Dialysis units of four academic medical centers. PARTICIPANTS: The study included 302 subjects (135 women and 167 men) on CAPD with four daily exchanges and a 2-L exchange volume. INTERVENTION: Measurement of urea and creatinine clearances (261 in women, 352 in men) by standard methods. Body water (the volume of distribution, V, for both urea and creatinine) was estimated by the Watson anthropometric formulas. MAIN OUTCOME MEASURES: Comparison of raw and normalized clearances between women and men. Urea clearance was normalized by V (Kt/Vur), while creatinine clearances was normalized by both V (Kt/Vcr) and body surface area (BSA) (Ccr). RESULTS: Mean values of weekly total (peritoneal plus renal) raw clearances were higher in men (urea clearance: women 67.1 L, men 77.4 L; Ccr: women 61.7 L, men 78.3 L). Raw renal clearances were higher in men, while raw peritoneal clearances were comparable. Mean weekly total Kt/Vur was higher in women (2.19 vs 1.94 in men), mean weekly total Kt/Vcr did not differ between the genders (women 2.01, men 1.95), while mean weekly Ccr was higher in men (73.0 vs 64.7 L/1.73 m2 in women). When clearances differed, the differences were significant at p < 0.001. Men had greater height and weight, while women had greater body mass index. On the average, V in men exceeded V in women by 31%, while BSA in men exceeded BSA in women by only 12%. CONCLUSIONS: Normalization of clearances by V creates relatively higher clearance values in women, while normalization by BSA creates relatively higher clearance values in men. Thus the normalization process may create artificial differences in the normalized clearances between genders. (+info)
Colicin E1 forms a dimer after urea-induced unfolding.
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Unfolding of the soluble colicin E1 channel peptide was examined with the use of urea as a denaturant; it was shown that it unfolds to an intermediate state in 8.5 M urea, equivalent to a dimeric species previously observed in 4 M guanidinium chloride. Single tryptophan residues, substituted into the peptide at various positions by site-directed mutagenesis, were employed as fluorescent probes of local unfolding. Unfolding profiles for specific sites within the peptide were obtained by quantifying the shifts in the fluorescence emission maxima of single tryptophan residues on unfolding and plotting them against urea concentration. Unfolding reported by tryptophan residues in the C-terminal region was not characteristic of complete peptide denaturation, as evidenced by the relatively blue-shifted values of the fluorescence emission maxima. Unfolding was also monitored by using CD spectroscopy and the fluorescent probe 2-(p-toluidinyl)-naphthalene 6-sulphonic acid; the results indicated that unfolding of helices is concomitant with the exposure of protein non-polar surface. Unfolding profiles were evaluated by non-linear least-squares curve fitting and calculation of the unfolding transition midpoint. The unfolding profiles of residues located in the N-terminal region of the peptide had lower transition midpoints than residues in the C-terminal portion. The results of unfolding analysis demonstrated that urea unfolds the peptide only partly to an intermediate state, because the C-terminal portion of the channel peptide retained significant structure in 8.5 M urea. Characterization of the peptide's global unfolding by size-exclusion HPLC revealed that the partly denatured structure that persists in 8.5 M urea is a dimer of two channel peptides, tightly associated by hydrophobic interactions. The presence of the dimerized species was confirmed by SDS/PAGE and intermolecular fluorescence resonance energy transfer. (+info)
Nitrogen intake and tumorigenesis in rats injected with 1,2-dimethylhydrazine.
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Tumor incidence was studied in 1,2-dimethylhydrazine (DMH) injected male rats assigned at weaning to isoenergetic casein-sucorse deits containing 7.5%, 15%, or 22.5% protein with or without 2.5% urea. Twenty rats fed each diet were given weekly intraperitoneal injections of DMH (15 mg/kg body weight/week) for the first 24 weeks and 20 were given saline. Of 96 DMH-injected rats necropsied after 28 weeks, 88 were necropsied during the 32nd or final week of the experiment. Adenocarcinomas of the small and large intestine were larger and significantly more numberous in rats fed 15% and 22.5% dietary protein. Keratin producing papillomas of the sebaceous glands of the external ear were observed first at 21 weeks in DMH-injected rats fed 22.5% protein. These were subsequently observed in some rats from all DMH-treated groups. As time progressed, the ear tumors increased in size and number in all groups but the greatest incidence was in the group fed 22.5% protein. No tumors were observed in saline-injected rats. Urea feeding did not increase the number of tumors nor cause changes in pH, urease activity or ammonia concentration of contents of the colon or cecum, or blood cholesterol. As dietary protein increased, cecal ammonia concentrations rose while both colon and cecal pH dropped. Portal blood urea and cholesterol reose as dietary protein was increased. DMH-treated rats had significantly higher concentrations of colon and cecal ammonia and lower blood cholesterol. Altough the rats fed 7.5% protein gained significantly less weight during 0 to 6 weeks of feeding, their weight gain was significantly higher during 6 to 26 weeks. No tumors were found in rats necropsied at 16 weeks. (+info)