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R De Smet

Publications and source records attributed to R De Smet.

At least 19 recordsLinked to original sources
PubMed2001

Kinetics of the protein-bound, lipophilic, uremic toxin p-cresol in healthy rats.

P-Cresol, a partially lipophilic and protein-bound compound is related to several biochemical alterations in uremia. Because p-cresol kinetics have never been studied, we investigated its kinetic behavior in rats. Results were compared with those obtained with creatinine, a water soluble, non-protein-bound uremic retention solute, which is currently used as a marker of uremic retention. Healthy rats were divided into 3 groups with comparable body weight: (1) a control group (n=6); (2) a group (n=7) which received an intravenous bolus of 3 mg p-cresol; and (3) a group (n=5) which received an intravenous bolus of 18 mg creatinine. Blood samples were collected at 0, 5, 30, 60, 120, 180 and 240 minutes after administration for the determination of p-cresol and creatinine. Urine was collected at 1-hour intervals. p-Cresol concentrations were assessed by HPLC. Pharmacokinetic parameters of p-cresol and creatinine were calculated from the serum concentration-time curves using non-compartmental analysis. Each compound showed a concentration at time point 5 min (p-cresol: 6.7 +/- 1.4 mg/L and creatinine: 141 +/- 12 mg/L) which was comparable with values observed in uremic patients; these concentrations decreased gradually towards min 240 (p-cresol: 0.6 +/- 0.3 mg/L and creatinine: 4 +/- 2 mg/L, p<0.05 vs. 5 min in both cases). No p-cresol was found in the serum of control rats and these rats showed no changes in serum concentration of creatinine. Urinary excretions were strikingly different (p-cresol: 23 +/- 10% and creatinine: 95 +/- 25% of the administered dose, p<0.05). The half-life of p-cresol was twice as long as that of creatinine (1.5 +/- 0.8 vs. 0.8 +/- 0.1 h, p<0.05). Total clearance (CLt) was much higher for p-cresol than for creatinine (23.2 +/- 4.5 vs. 8.1 +/- 0.4 mL/min/kg, p<0.01); renal clearance (CLr), however, was substantially lower for p-cresol (4.8 +/- 2.0 vs. 8.2 +/- 1.9 mL/min/kg, p<0.05). Whereas CLt and CLr were similar for creatinine, CLt of p-cresol largely exceeded its CLr (p<0.05). The volume of distribution (Vd) was also much larger for p-cresol than for creatinine (2.9 +/- 1.4 vs. 0.6 +/- 0.1 L/kg, p<0.01). After injection of p-cresol, an additional chromatographic peak appeared in serum and in urine samples. Although at min 240 serum concentration of p-cresol had decreased to 10% of the peak value, only 23% of the administered amount was excreted in the urine and the CLr was +/- 50% lower compared to that of creatinine. Non-renal clearance and Vd of p-cresol were, however, substantially larger. These data may be of value to explain the different behavior of p-cresol in renal failure and dialysis, compared to creatinine.

Animals↗
PubMed2001

Protein-bound uremic solutes: the forgotten toxins.

The present concept of dialysis focuses mainly on the removal of small water-soluble compounds, and also, the currently applied kinetic parameters of dialysis adequacy are based on the behavior of water-soluble compounds. Nevertheless, many of the currently known biological effects in uremia are attributable to compounds with different physicochemical characteristics, and among these, protein-bound solutes play an important role. In this article, we review the characteristics and consequences of changes in protein binding in uremia, as well as the toxicity of the protein-bound uremic solutes 3-carboxy-4-methyl-5-propyl-2-furanpropionic acid (CMPF), indoxyl sulfate, hippuric acid, homocysteine, and p-cresol. Starting from the example of p-cresol, we then summarize the impact of protein-binding on dialytic removal, whereby it is concluded that this removal is largely hampered by this protein-binding compared with that of classic markers such as urea and creatinine. Alternative removal strategies, such as strategies to modify intestinal generation or absorption, are considered.

Animals↗
PubMed2001

Uremic toxins and peritoneal dialysis.

Uremic toxicity is related in part to the accumulation of toxic substances, the nature of which has only partly been characterized. Because of the use of a highly permeable membrane and better preservation of the residual renal function, it could be anticipated that some of these uremic toxins are more efficiently cleared across the peritoneal membrane, and that the plasma and tissue levels of these compounds are lower than in hemodialysis patients. This article analyzes the generation and removal of several uremic toxins in peritoneal dialysis patients. The following uremic toxins are discussed: beta2-microglobulin, advanced glycation end products, advanced oxidation protein products, granulocyte inhibitory proteins, p-Cresol, and hyperhomocysteinemia. Some recent studies are reviewed suggesting that uremic toxins are involved in the progression of renal failure and are at least partially removed by peritoneal dialysis. We conclude that, although the plasma levels of some of these compounds are lower in peritoneal dialysis versus hemodialysis patients, it does not mean that the peritoneal dialysis patient is "better" protected against the numerous disturbances caused by these toxins.

Animals↗
PubMed2001

The anti-proliferative effect of calcitriol on HL-60 cells is neutralized by uraemic biological fluids.

BACKGROUND: It has been demonstrated that uraemic serum/ultrafiltrate inhibits cell-mediated immune response in vitro, and that it suppresses calcitriol synthesis and its biological actions. METHODS: In the present in vitro study, the effect of calcitriol, uraemic ultrafiltrate (UUF) and a combination of both on the human promyelocytic leukaemia cell line, HL-60, was studied by evaluating bromodeoxyuridine (BrdU) incorporation into the DNA, luminol-amplified chemiluminescence (CL) production, expression of CD14, and levels of vitamin D receptor mRNA (VDR mRNA) and CD14 mRNA. RESULTS: The ability of calcitriol to block cell proliferation (37.4+/-5.4 to 30.5+/-5.6% cells incorporating BrdU, P:<0.01) was neutralized when UUF was applied together with calcitriol (53.4+/-21.3% cells incorporating BrdU, P:<0.01 vs calcitriol alone). Similarly to what was observed for BrdU incorporation, the CL production of HL-60 cells was enhanced in the presence of calcitriol (20126+/-10154 to 61528+/-24021 cpm, P:<0.01), and was suppressed again in the presence of calcitriol and UUF (20916+/-12075 cpm, P:<0.01 vs calcitriol alone); finally UUF also inhibited the calcitriol-induced CD14 expression (71.1+/-11.2 to 54.9+/-17.7% CD14 positive cells, P:<0.05). On the other hand, the calcitriol-induced CD14 mRNA levels were not significantly different in the presence of calcitriol and UUF compared to calcitriol alone. This points to an inhibition by UUF at a post-transcriptional level. Similar data were found for VDR mRNA levels. UUF was fractionated by HPLC in four fractions, hydrophilic uraemic solutes being eluted first (F1) and hydrophobic solutes being eluted last (F4); fractions 1, 2 and 3 simultaneously affected both BrdU incorporation and CL production in a significant way. CONCLUSIONS: It is concluded that UUF contains factors that impair calcitriol-activated function of HL-60 cells. Hence, the differentiation and immune response of these promyelocytic leukaemia cells, as induced by the supplementation of calcitriol, is neutralized in the presence of uraemic biological fluids. This may be of relevance for the propensity to infection and malignancy of the uraemic patient.

Biological Factors↗
PubMed2001

Heparin-induced release of protein-bound solutes during hemodialysis is an in vitro artifact.

BACKGROUND: Several studies have pointed to a release of drugs or protein-bound solutes from their binding sites during heparinization. The effect is attributed to the metabolism of triglycerides to free fatty acids (FFAs), which compete with drugs for protein binding sites. This study evaluated the impact of intradialytic heparin on the free concentration of the uremic toxin p-cresol and on FFAS: METHODS: Blood samples from hemodialysis (HD) patients, before and during HD, were collected with selected anticoagulation strategies. We assessed the effects of standing time, temperature, pH, and the addition of a lipase inhibitor, tetrahydrolipstatin (THL) to blood samples on the free p-cresol concentration. p-Cresol was analyzed by HPLC with fluorescence detection. We measured FFAs by gas chromatography, and the free fractions of added valproic acid and phenytoin were evaluated by fluorescence polarization immunoassay. RESULTS: In blood samples (n = 22) not submitted to a specific treatment, free p-cresol increased from 9.9 +/- 5.1 to 31.9 +/- 22.3 micromol/L after 30 min of heparin HD (P < 0.001) and correlated significantly with FFAs (r = 0.80; P = 0.002; n = 12). There was no increase in free p-cresol during heparin-free HD (n = 6) and trisodium citrate HD (n = 9). In addition, p-cresol in ultrafiltrates (n = 3) did not correspond to the free p-cresol in heparinized blood, suggesting that the increase in free p-cresol was artifactual. The release of p-cresol in the test tube was enhanced by standing time (n = 6), sample temperature (n = 6), and alkaline pH (n = 6). Inhibition of lipase activity with THL prevented the increase of FFAs (n = 6) and the release of free p-cresol during HD (n = 22). These results were corroborated by the study of the free fraction of valproic acid (n = 6) and phenytoin (n = 6). CONCLUSIONS: The free concentrations of protein-bound solutes in plasma of heparinized patients are influenced by external factors that alter the lipase activity in the test tube. The free fraction does not increase during HD when lipase activity is neutralized at the time of blood sampling, so that previously reported increases are probably artifacts.

Aged↗
PubMed2001

Advanced glycation end products: specific fluorescence changes of pentosidine-like compounds during short daily hemodialysis.

BACKGROUND: Advanced glycation end products (AGE) accumulate in uremia and represent an important etiopathogenetic cause of morbidity in dialyzed patients. Conventional hemodialysis treatment seems to be ineffective in lowering AGE levels. We wished to investigate whether daily hemodialysis (DHD), a treatment that seems to result in better clinical condition in end-stage renal disease patients, is effective in the reduction of these compounds. METHODS: We evaluated 10 non-diabetic patients on standard hemodialysis (SHD = 3 x 4 h/week) for more than 6 months by a crossover study. These patients were assigned randomly to 6 months of DHD (6 x 2 h/week) or 6 months of SHD. Then, they were switched to 6 months of the alternative treatment. At the end of these two periods, we studied pentosidine-like AGE compounds by measuring the total fluorescence at a wavelength characteristic for these substances: Ex: 335nm/Em:385nm; we also measured protein-linked pentosidine at the same time points. Finally, we determined the AGE-related total fluorescence in the deproteinized serum of 13 uremic patients on peritoneal dialysis (CAPD) and of 10 healthy controls. RESULTS: Pre-HD AGE-related total fluorescence obtained after 6 months of DHD was significantly lower than that obtained with standard HD (DHD = 201.3 +/- 36.4 AU/ml vs. SHD = 267.5 +/- 141.4 AU/ml, p = 0.03). The extraction rate per minute of dialysis was slightly, but not significantly higher during DHD than SHD (0.29 +/- 0.11% vs. 0.23 +/- 0.04, p = 0.07). AGE-related total fluorescence pre-HD values in patients treated by SHD and DHD were about 20-fold higher than in control subjects. They did not differ from CAPD patients. The pre-dialysis level of protein-linked pentosidine was significantly lower in DHD than in SHD (DHD = 16.12 +/- 4.71 pmol/mg protein, SHD = 22.64 +/- 6.86 pmol/mg protein, p < 0.01). CONCLUSIONS: DHD showed a reduction in AGE-related total fluorescence, although the mean value remained higher than in control subjects. DHD is also accompanied by a decrease in protein-linked pentosidine.

Arginine↗
PubMed2000

Vitamin E-bonded cellulose membrane and hemodialysis bioincompatibility: absence of an acute benefit on expression of leukocyte surface molecules.

Dialysis with unmodified cellulose membranes is associated with such bioincompatibility phenomena as leukopenia, increased expression of adhesion molecules on leukocytes, and release of reactive oxygen species. Dialysis biocompatibility can be improved by modifications in the structure of the cellulose membrane to diminish leukocyte activation and/or protect against the released free oxygen radicals. Excebrane (Terumo Corp, Tokyo, Japan) is a vitamin E-modified cellulose membrane. In the present study, the effect of dialysis with Excebrane membranes on granulocyte and monocyte counts; CD11b, CD11c, and CD45 expression on the surface of granulocytes; and CD14 expression on monocytes was evaluated and compared with low-flux polysulfone membranes. Fifteen minutes after the start of dialysis, granulocytopenia and monocytopenia were more pronounced with the Excebrane membrane compared with polysulfone. The increase in basal expression of CD11b and CD45 on circulating granulocytes was more pronounced during dialysis with Excebrane than polysulfone membranes. Regarding the increased expression on in vitro stimulation with phorbol myristate acetate, blunted upregulation was obtained during dialysis using Excebrane membranes for CD11c and CD45 expression on granulocytes and CD14 expression on monocytes. In conclusion, such indices of membrane bioincompatibility as leukocyte counts and expression of leukocyte surface molecules show more profound alterations with Excebrane than the standard low-flux polysulfone membrane in both basal and in vitro activated states.

Biocompatible Materials↗
PubMed2000

Intradialytic removal of protein-bound uraemic toxins: role of solute characteristics and of dialyser membrane.

BACKGROUND: The efficiency of dialysis membranes is generally evaluated by assessing their capacity to remove small, water-soluble and non-protein-bound reference markers such as urea or creatinine. However, recent data suggest that protein-bound and/or lipophilic substances might be responsible for biochemical alterations characterizing the uraemic syndrome. METHODS: In the present study, the total concentrations of four uraemic retention compounds (indoxyl sulphate, hippuric acid, 3-carboxy-4-methyl-5-propyl-2-furanpropionic acid (CMPF) and p-cresol) and of tryptophan, the only protein-bound amino acid and a precursor of indoxyl sulphate, were compared with those of urea and creatinine in pre- and post-dialysis serum and in dialysate of 10 patients; two high-flux (HF) membranes (cellulose triacetate (CTA) and polysulphone (PS)) and a low-flux polysulphone (LFPS) membrane were compared in a crossover design, using HPLC. RESULTS: Except for hippuric acid (67.3+/-17.5% decrease), major differences were found in the percentage removal of the classical uraemic markers on one hand (creatinine 66.6+/-7.0% and urea 75.5+/-5.8% decrease) and the studied protein-bound and/or lipophilic substances on the other (indoxyl sulphate, 35.4+/-15.3% and p-cresol 29.0+/-14.2% decrease; tryptophan, 27.5+/-40.3%, and CMPF, 22.4+/-17.5% increase; P<0.01 vs urea and creatinine in all cases). Hippuric acid removal was more pronounced than that of the remaining protein-bound compounds (P<0. 01). After correction for haemoconcentration, per cent increase of tryptophan and CMPF was less substantial, while per cent negative changes for the remaining compounds became more important. There was a correlation between creatinine and urea per cent removal at min 240 (r=0.51, P<0.01), but all the other compounds showed no significant correlation with either of these two. The three membranes were similar regarding the changes of total solute concentrations from the start to the end of dialysis. CONCLUSIONS: Urea and creatinine are far more efficiently removed than the other compounds under study, except for hippuric acid. There are no striking differences between the HF membranes. Moreover, compared with the LF membrane these HF membranes do not appear to be superior in removing the studied compounds.

Aged↗
PubMed2000

Effect of regional citrate anticoagulation on leukopenia, complement activation, and expression of leukocyte surface molecules during hemodialysis with unmodified cellulose membranes.

BACKGROUND: Dialysis with complement-activating membranes is associated with leukopenia, which is related to an increased expression of adhesion molecules on leukocytes. Citrate chelates calcium and has been claimed to attenuate leukopenia. METHODS: In this study, the effects of citrate anticoagulation on leukocyte and granulocyte counts, complement activation, and the expression of CD11b, CD11c, and CD45 on the surface of granulocytes were evaluated during hemodialysis with unmodified cellulose membranes. Standard heparin was compared to citrate in three different schedules: citrate was infused to obtain a concentration of either 7 or 10 mmol/l blood. CaCl(2) was administered into the dialyzer outlet at 8. 25 mmol Ca(2+)/h (citrate 10 mmol/l) or at 11 mmol Ca(2+)/h (citrate 7 and 10 mmol/l) to reconstitute the calcium levels in the blood returning to the patient. RESULTS: The use of citrate at a high concentration (10 mmol/l) was associated with a blunted upregulation of CD11b, both at the inlet and at the outlet bloodline; for CD11c a reduced upregulation was observed on granulocytes harvested from the inlet bloodline. No effects of citrate were observed on leukopenia, granulocytopenia, or complement activation. A positive correlation between the decrease in systemic ionized Ca(2+) concentration and the increase in CD11b and CD11c expression was found. CONCLUSION: Citrate/CaCl(2) administration affects leukocyte adhesion molecule expression in a dose-dependent way; however, no significant effect could be demonstrated on leukopenia and complement activation.

Adult↗
PubMed1999

Study by means of high-performance liquid chromatography of solutes that decrease theophylline/protein binding in the serum of uremic patients.

Substantial changes in protein binding of drugs occur during the progression of renal insufficiency. Protein-bound uremic solutes play a role in the inhibition of drug protein binding. We previously demonstrated that hippuric acid in uremic ultrafiltrate was an inhibitor of the theophylline protein binding. The present study was undertaken to extend the yield of protein-bound uremic solutes by displacing ligands in uremic serum from their binding sites by five deproteinization methods. The inhibitory effect on theophylline protein binding of the deproteinized uremic serum was higher than with ultrafiltrate (p < 0.05). The influence of 30 semi-preparative HPLC fractions from deproteinized uremic serum on the theophylline protein binding was evaluated to identify the responsible compounds and to compare their relative individual impact. The theophylline protein binding was calculated as a percentage (bound versus total). The most important decrease of the protein binding was observed in HPLC fractions 6, 10 to 13, 15 and 28 with protein binding of: 61.5 +/- 10.8, 64.5 +/- 7.6, 60.9 +/- 10.1, 47.5 +/- 3.3, 60.0 +/- 6.7, 60.7 +/- 6.3 and 61.3 +/- 6.9%, respectively versus 69.1 +/- 2.4% for control serum (p < 0.05). The responsible compounds were characterized in the fractions by co-elution: 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid (CMPF), indole-3-acetic acid, indoxyl sulfate, hippuric acid, p-hydroxyhippuric acid and tryptophan. Their concentration was determined by analytical HPLC and a solution containing these compounds at the same concentration as in deproteinized uremic serum was composed. This solution was added to control serum and decreased the theophylline protein binding from 69.0 +/- 4.4% to 61.3 +/- 1.3%, which was less important than in genuine uremic serum (44.4 +/- 3.8%, p < 0.05). Dose-response curves with the characterized compounds revealed that the most important role in binding inhibition could be attributed to hippuric acid and CMPF. Our data suggests that the yield of protein binding inhibiting compounds is more important with deproteinized uremic serum than with uremic ultrafiltrate. The identified uremic compounds are not entirely representative for the decreased protein binding of theophylline, indicating that additional factors than those identified in this study affect the protein binding as well.

Blood Proteins↗
PubMed1999

Uremic ultrafiltrate inhibits platelet-activating factor synthesis.

BACKGROUND: Several studies have suggested that uremic toxins may adversely affect phagocytic leukocytes of chronic renal failure patients. Platelet-activating factor (PAF) is produced by phagocytic leukocytes and is a potent mediator of inflammation which is produced by leukocytes upon appropriate stimulation. METHODS: We added uremic or normal ultrafiltrate, ultrafiltrate fractionated by reverse phase HPLC or compounds eluting at the same retention time as the fractionated ultrafiltrate, to normal leukocytes. Complement-coated baker's yeast spores were added to stimulate phagocytosis. Total PAF was purified by thin layer chromatography and quantified by bioassay on rabbit platelets. The activities of two enzymes involved in the synthesis of PAF, phospholipase A2 (PLA2) and acetyltransferase, were measured in the presence of fractionated ultrafiltrate. RESULTS: Ultrafiltrate from both healthy and uremic subjects inhibited PAF synthesis, but the inhibitory effect was more substantial for uremic subjects. Ultrafiltrate fractionated by HPLC showed high PAF inhibition for late eluting hydrophobic fractions. Addition of phenol or p-cresol, two uremic toxins with similar elution pattern as the late fractions, also inhibited PAF synthesis. The activity of PLA2 and acetyltransferase was decreased in the presence of uremic ultrafiltrate. CONCLUSIONS: We observed that uremic ultrafiltrate inhibits PAF synthesis upon stimulation with complement coated baker's yeast spores. The decrease in total PAF synthesis appears to be associated with an inhibition of phospholipase A2 and acetyltransferase activity, enzymes involved in the remodelling pathway for PAF synthesis.

Adult↗
PubMed1998

A sensitive HPLC method for the quantification of free and total p-cresol in patients with chronic renal failure.

Para-cresol (4-methylphenol) is a volatile phenolic compound which is retained in chronic renal failure. Several recent studies suggest that p-cresol interferes with various biochemical and physiological functions at concentrations currently observed in uremia. Only a few methods are available for the determination of p-cresol concentration in serum. In addition, these methods have only been used for the determination of total p-cresol. In particular, the evolution of free (non-protein bound) p-cresol is of concern, because conceivably this is the biologically active fraction. The concentration of free p-cresol, is, however, markedly lower than that of total p-cresol, in view of its important protein binding. We report a method enabling the measurement of total and free p-cresol concentration in serum of healthy controls and uremic patients. Deproteinization, extraction and HPLC procedure are efficient, without interference of other protein bound ligands and/or precursors of p-cresol or phenol. By means of spiking experiments, the measurement of the UV absorbance over the 200-400 nm wavelength range, and capillary gas chromatography-mass spectrometry, the considered compound is identified as p-cresol. With a fluorescence detection at 284/310 nm as extinction/emission wavelengths the detection limit of p-cresol is 1.3 micromol/l (0.14 microg/ml). Recovery of added p-cresol to normal serum is 95.4+/-4.1%. For free p-cresol and total p-cresol determinations, intra-assay and day-to-day variation co-efficients are 3.2%, 4.2%, 6.9% and 7.3%, respectively. Compared to healthy controls, the serum p-cresol levels are 7-10 times higher in continuous ambulatory peritoneal dialysis patients (CAPD), uremic outpatients, and hemodialysis patients: 8.6+/-3.0 vs. 62.0+/-19.5, 87.8+/-31.7 and 88.7+/-49.3 micromol/l (0.93+/-0.32 vs. 6.70+/-2.11, 9.49+/-3.43, and 9.60+/-5.30 microg/ml) (p<0.05), respectively. The difference is even more important if free p-cresol is considered. This corresponds to a decreased protein binding in uremic patients. We conclude that the present method allows an accurate measurement of both total and free p-cresol, and that the measured concentrations in uremia are in the range which may cause biochemical alterations.

Artifacts↗
PubMed1998

Citrate anticoagulation does not correct cuprophane bioincompatibility as evaluated by the expression of leukocyte surface molecules.

BACKGROUND: Citrate, used for the anticoagulation of the extracorporeal dialysis circuit, reduces ionized calcium by chelation and has been claimed to attenuate dialyser membrane bioincompatibility. Dialysis with complement-activating cuprophane membranes is associated with leukopenia which has been related to an increase in adhesion molecule expression on the surface of circulating leukocytes. METHODS: The effect of citrate anticoagulation on the expression of CD11b, CD11c and CD45 on the surface of granulocytes and CD14 on monocytes during haemodialysis with cuprophane membranes, was evaluated by flow cytometric analysis. A comparison of standard heparin vs citrate was performed in 14 chronic haemodialysis patients. During citrate anticoagulation a calcium-free dialysate was used and citrate was infused to obtain a concentration of 4.3 mmol/l blood. The unchallenged 'baseline state' expression of the surface molecules and the increase after ex vivo stimulation with phorbol 12-myristate 13-acetate (delta-PMA) or formyl-methionyl-leucyl-phenylalanine (delta-fMLP) was studied. RESULTS: With heparin, as well as with citrate, a sharp fall in granulocyte and monocyte count was observed after 15 min of dialysis, followed by a recovery at the end of the session. The expression of CD11b, CD11c and CD45 on granulocytes increased markedly during cuprophane dialysis with a peak at 15 min; there were no differences in response between heparin and citrate anticoagulation. Delta-PMA and delta-fMLP for CD45, CD11c and CD14 showed a decrease during cuprophane dialysis vs t0; again there were no differences between heparin and citrate. CONCLUSION: We conclude that the use of citrate was not associated with reduced leukocyte activation as measured by the expression of surface molecules during cuprophane dialysis and that no effect on dialysis leukocytopenia could be registered.

Aged↗