Inhibition of phagocytosis by a middle molecular fraction from ultrafiltrate.

The in vitro phagocytic activity of normal human blood was maximally inhibited by an uremic toxin isolated by Sephadex G 15 column chromatography from the ultrafiltrate obtained during a sequential hemodiafiltration procedure in an anephric patient. The phagocytic activity of the blood was studied by measuring the labelled CO2 production from glucose metabolism during the phagocytosis of latex, zymosan and inulin; phagocytosis was not influenced by urea levels of 4 g/l and creatinine levels of 15 mg/100 ml, whereas it was inhibited for 59% by uremic serum and for 90% by a D fraction. The D fraction blocking phagocytosis is of the so-called middle molecular weight range; the presumed molecular weight is between 113 and 1029; the elution volume Ve is 25.28/ml and the available distribution coefficient KAV 0.52.

Phagocyte metabolic activity during hemodialysis with different dialyzers not affecting the number of circulating phagocytes.

Overall leukocyte counts decrease during certain forms of hemodialysis, but little information is available on the intradialytic evolution of phagocytic metabolic function, especially during dialysis with dialyzers not affecting the number of circulating phagocytes. This study evaluated the phagocytic capacity of granulocytes and monocytes to generate CO2 out of glucose under basic unchallenged conditions and after stimulation with latex or zymosan, before and after 15, 60 and 240 minutes of dialysis with reused cuprophan, AN69S, polysulphone, polymethylmethacrylate and hemophan hemodialyzers. Phagocytic metabolic function was assessed in whole blood on the basis of 14CO2-production from labelled glucose during the phagocytic process. There were no changes in basic unchallenged CO2-production with any of the dialyzers. Reactivity to latex and zymosan, expressed per number of phagocytes, showed no decrease, irrespective of the membrane type. For polymethylmethacrylate and reused cuprophan, a slight but significant increase in metabolic reactivity was observed in response to latex and zymosan. The test employed may give a screening picture of the phagocytic reaction to contact with dialyzers and membranes and thus of their degree of biocompatibility towards the phagocyte system.

Isotachophoretic pattern of LDH inhibiting fractions obtained from uremic ultrafiltrate by gelchromatography.

Uremic ultrafiltrate was fractioned by chromatography on Sephadex G15; two fractions were highly inhibiting the total lactate dehydrogenase activity in rat kidney homogenate. The inhibiting fractions were eluates number 10 and 11 with an elution volume of respectively 17.4 ml and 19.3 ml and a Kav of 0.17 and 0.25. Both fractions presented a different pattern on analysis by analytical isotachophoresis.

Cefodizime: a new cephalosporin with apparent immune-stimulating properties in chronic renal failure.

In vitro experiments suggest that cefodizime, a new cephalosporin, causes an increase in phagocytic capacity. We therefore evaluated the effect of cefodizime on the phagocytic system in haemodialysis patients by an estimation of the 14CO2 production during glucose metabolisation by phagocytic cells, in the resting state, and after zymosan and latex. The production of 14CO2 after latex increased in five of six patients (mean +/- SD: from 17,932 +/- 11,859 before to 21,183 +/- 7849 d.p.m. at the end of treatment). The corresponding data after zymosan were 48,381 +/- 24,891 and 70,176 +/- 15,140 d.p.m. The improved 14CO2 production after stimulation persisted for 2 further weeks. These results suggest a stimulation in vivo of the depressed phagocytic system of the uraemic patient by cefodizime.

Influence of human urine on phagocytosis.

Human urine may inhibit or activate the phagocytosis by normal human blood of latex, zymosan and inuline. No specific difference was found between the urine of normals and patients with moderate, severe or end stage renal failure. The inhibiting effect was not due to urea or creatinine; the assumption is made that a middle molecular fraction D, which can be isolated by Sephadex G 15 columnchromatography from uraemic ultrafiltrate, is responsible for the observed inhibition. Urines of patients with significant bacteriuria were more frequently activating.

Drug protein binding in chronic renal failure: evaluation of nine drugs.

In this study, changes of protein binding of nine drugs were evaluated. In addition, theophylline and phenytoin, the two drugs with the most substantial and progressive decrease in protein binding, were further studied by high performance liquid chromatography (HPLC)-fractions of ultrafiltrate of normal and uremic serum, in an attempt to identify substances causing drug protein binding inhibition. There was a marked decline of the protein binding of theophylline, phenytoin and methotrexate (dialyzed patients vs. normals: -20.1, -16.0 and -15.1%, respectively). There was a rise in the protein binding of propranolol, cimetidine and clonidine. The changes observed for diazepam, prazosin and imipramine were less marked. For phenytoin, theophylline, methotrexate and diazepam, protein binding was inversely correlated to the serum creatinine (r = 0.87, 0.80, 0.79 and 0.67, P less than 0.001), and a less pronounced but still significant positive correlation was found for clonidine (r = 0.46, P less than 0.01). Ultrafiltrate, obtained during a hemofiltration session, inhibited protein binding of theophylline and phenytoin in a dose dependent way. After separation of this ultrafiltrate by HPLC, it appeared that for both theophylline and phenytoin at least a part of this inhibitory activity corresponded to the elution zone of hippuric acid. For theophylline two other inhibitory zones were further recognized: one corresponding to the elution zone of NaCl and one in which the responsible substance remained unidentified. Hippuric acid in solution inhibited protein binding of theophylline and phenytoin in a dose dependent way. In conclusion, protein binding of several drugs currently used in renal failure is affected in parallel with renal function, which might affect the therapeutic effectiveness of the drugs.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibiotics and energy delivery to the phagocytosis-associated respiratory burst in chronic hemodialysis patients: a comparison of cefodizime and cotrimoxazole.

Twenty-three stabilized chronic uremic patients with no active or recent infection were treated for 10 days with either cefodizime (5 x 2 g intravenously, n = 10) or cotrimoxazole (960 mg orally b.i.d., n = 8) in order to evaluate the effects on the depressed polymorphonuclear metabolic response to phagocytic challenge; a separate group of 5 patients received placebo. Ex vivo evaluation in whole blood of energy delivery to the phagocytosis-associated respiratory burst activity in response to latex and zymosan challenge was determined by measuring hexose-monophosphate shunt NAD(P)H-oxidase-related glycolytic activity. Cefodizime induced a statistically significant increase in the baseline-depressed glycolytic response for both latex and zymosan challenge, in contrast to cotrimoxazole and placebo. Depressed phagocytosis-related metabolic function in hemodialyzed patients was stimulated by cefodizime in recommended therapeutic doses but not by cotrimoxazole, the effect persisting for at least 2 weeks after the end of treatment.

Mechanisms of uremic inhibition of phagocyte reactive species production: characterization of the role of p-cresol.

It is generally recognized that the uremic syndrome results in a depression of immune function, but the uremic solutes responsible remain largely unidentified. In this study, the effect of 18 known uremic retention solutes, including urea and creatinine, on hexose monophosphate shunt (HMS)-dependent glucose-1-C14 utilization (G1C-U), chemiluminescence production (CL-P) and flow cytometric parameters (FCP) of respiratory burst and phagocytosis were evaluated in granulocytes and/or monocytes. Among the compounds studied, only p-cresol depressed whole blood respiratory burst reactivity (G1C-U, CL-P) dose dependently at concentrations currently encountered in end-stage renal disease (ESRD) (P < 0.05 from 5 micrograms/ml on). The effect of p-cresol was enhanced by increasing incubation times from 10 to 120 minutes. HMS activity of isolated packed erythrocytes remained unaffected. FCP of respiratory burst activity (Bursttest, expressed as log fluorescence units, LFU) revealed a marked depression in the presence of p-cresol (from 700 +/- 167 to 291 +/- 128 LFU for granulocytes, from 278 +/- 102 to 146 +/- 52 LFU for monocytes, P < 0.01), whereas particle ingestion (Phagotest) remained unaffected. Cell-free myeloperoxidase activity was also markedly depressed in the presence of p-cresol. Polarity based HPLC-elution of a standard solution containing all the solutes studied, using a gradient from 100% formic acid to 100% methanol during 60 minutes, revealed elution of p-cresol after 46.6 minutes, pointing to its relative hydrophobicity. Conjugation of p-cresol to p-cresylsulfate anihilated the depressive effect of p-cresol on granulocyte function, and at the same time caused a shift in HPLC-elution pattern to a less lipophilic range.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Uraemic toxic retention solutes depress polymorphonuclear response to phagocytosis.

Previous studies from our laboratory have demonstrated that the activity of the hexose monophosphate shunt (HMS) pathway in phagocytosis-related respiratory burst is disturbed in end-stage renal disease. To determine whether uraemic solute retention is responsible for this defect the HMS-path was evaluated by measurements of glucose-1-C14 utilization and determination of 14CO2 production in polymorphonuclear cells (PMNLs), suspended in normal plasma or uraemic biological fluids. Normal PMNLs, while suspended in normal or uraemic plasma, were stimulated with either latex, zymosan or Staph. aureus; CO2 generation (measured as DPM/10(3) PMNL, normal versus uraemic plasma) was depressed in uraemic plasma in response to latex (from 43 +/- 5 to 20 +/- 3), zymosan (from 72 +/- 8 to 47 +/- 4) (P < 0.01), and Staph aureus (from 73 +/- 17 to 47 +/- 8 DPM/10(3) PMNL) (P < 0.05). The degree of inhibition was similar for each stimulus. To characterize the substances responsible for this defect we fractionated uraemic plasma ultrafiltrate by polarity-based semipreparative C18 reversed phase HPLC and found a decreased response to Staph. aureus in the presence of fraction 2 (from 102 +/- 13 to 23 +/- 10 DPM/10(3) PMNL, P < 0.05), and in fractions 8 and 11 (lowest value in fraction 8, 54 +/- 14 DPM/10(3) PMNL, P < 0.05 versus control). The pattern of HPLC elution on a gradient from 100% formiate (pH 4.0) to 100% methanol indicates that there are at least two chemically distinguishable groups of compounds, one hydrophilic (in fraction 2), and one lipophilic (in fractions 8 and 11). We conclude that uraemic biological fluids contain factors that inhibit HMS activity related to phagocytosis, and that at least two groups of components with different characteristics are involved.

Depressed phagocytosis in hemodialyzed patients: in vivo and in vitro mechanisms.

Infection is a frequent complication and the major cause of death among end-stage renal patients. Polymorphonuclear phagocytes (PMNL) are important in host defense mainly because of bacterial destruction by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-related free radical production following phagocytosis. In this study, hexose monophosphate pathway glycolytic activity, delivering energy to NADPH oxidase, is evaluated in vivo and in vitro, in healthy controls and in dialyzed renal failure patients. Our results show a marked parallel and correlated inhibition in the response to three stimuli for phagocytic activity (Staphylococcus aureus, formyl-methionine-leucine-phenylalanine, phorbol myristic acid) in predialysis samples. These data point to a main suppression of metabolic pathways, possibly beyond protein kinase C. This response is further suppressed at the 15th minute of cuprophane dialysis, for all stimuli studied (-40 to -94%; p < 0.001) except PMA. PMNL response remains intact during dialysis with non-complement-activating dialyzers. In vitro experiments confirm decreased PMNL glycolytic activity after the suspension of cuprophane fragments in normal whole blood. We conclude that polymorphonuclear cell energy delivery to NADPH oxidase is impaired in patients with end-stage renal failure. The impaired response against various stimuli is different in predialysis blood samples compared to samples collected during cuprophane dialysis, and may be related to two different conditions. These events probably contribute to the acquired immune suppression of uremia and the high incidence of infection among dialysis patients.

Disturbed host defense in peritoneal cavity during CAPD: characterization of responsible factors in dwell fluid.

In this study, the factors in overnight dwell fluid (8 to 10 hr dwell) depressing granulocyte (GC) NAD(P)H-oxidase dependent radical species production are characterized. At present, most studies have essentially focused on fresh, unspent dialysate and on peritoneal macrophages. The response to Staphylococcus aureus (Staph A) was dose-dependently depressed for both GC CO2 production (from 91.3 +/- 8.4 to 9.0 +/- 1.5 dpm/10(3) GC, P < 0.01) and chemiluminescence (CL) (peak from 7.3 +/- 0.8 to 1.6 +/- 0.8 cps x 10(3)/GC, P < 0.01). Stimulation with formyl-methionine-leucine-phenylalanine (f-MLP), phorbol myristic acid (PMA), Staphylococcus epidermidis (Staph Epi), E. coli, latex and zymosan revealed a parallel depression, pointing to an intrinsic metabolic defect, rather than failure of particle ingestion. The addition of glucose to the normal cell medium to obtain the same concentration as in the CAPD effluent (2.9 +/- 0.3 mg/dl) depressed function but not to the same extent as the genuine PD effluent. Opsonization of Staph A and E. coli induced a partial correction. No effect of pH or osmolality was observed. HPLC fractionation of CAPD effluent on a polarity based gradient revealed an elution of depressive factors in hydrophobic fractions with a nadir in F7 and F12. Analysis of the elution pattern of various uremic solutes revealed elution in F12 of p-cresol, a solute with known inhibitory effect on GC function. These events may be related to recent peritonitis (CL in response to Staph A 0.3 +/- 0.1 in effluent of 6 patients with recent peritonitis versus 2.6 +/- 0.8 cps x 10(3)/GC in 12 patients without recent peritonitis (P < 0.01). We conclude that the GC response is depressed in the presence of CAPD effluent due to excess glucose, lack of opsonization, and uremic solutes of which p-cresol is one of the responsible compounds.