Uremic Plasma Research Articles

Decreased binding of drugs and dyes to plasma proteins from rats with acute renal failure: effects of ureter ligation and intramuscular injection of glycerol.

1 The decreased binding of drugs and dyes to plasma proteins from male and female rats with acute renal failure has been investigated using equilibrium dialysis at 37 degrees C. 2 Acute renal failure induced by bilateral ligation of the ureters produced a greater than threefold increase in the unbound fraction of o-methyl red relative to normal rat plasma. Unbound dye concentration in plasma from sham-operated control rats was also significantly increased but to a lesser extent. 3 Glycerol-induced acute renal failure produced a significant increase in the unbound fractions of o-methyl red, methyl orange, bromocresol green (BCG), 2-(4'-hydroxybenzeneazo) benzoic acid (HABA), phenytoin and salicylic acid. A marginally significant increase in unbound warfarin concentration was observed. 4 Glycerol-induced renal failure had no effect on total plasma protein concentration and experiments with o-methyl red and salicylic acid indicated that a direct effect of glycerol was not responsible for the diminution of binding. 5 Glycerol-induced acute renal failure, which produced decreases in drug and dye binding similar to those reported for human uraemic plasma, provides a convenient non-surgical animal model for the investigation of this phenomenon.

Vascular Prostacyclin and Plasminogen Activator Activity in Experimental and Clinical Conditions of Disturbed Haemostasis or Thrombosis

In several experimental and clinical conditions associated with thrombotic tendency, or complicated by thrombotic episodes, either prostacyclin (which inhibits platelet aggregation) or plasminogen activator (which promotes fibrinolysis), or both, appear to be decreased in the vessel wall. In other conditions, however, either activity may not change or even be increased. Possibly, vascular damage is followed by an early stimulation of both activities (a defence mechanism?) which may be subsequently reduced or exhausted. While the role of vascular plasminogen activator in haemorrhagic conditions is apparently unknown, prostacyclin activity appears to be markedly enhanced both in experimental animals and in patients with uraemia and bleeding complications. There is a suggestive evidence that uraemic plasma powerfully stimulates vascular prostacyclin generation.

URAEMIC TOXINS OF HIGH MOLECULAR WEIGHT INHIBITING HUMAN MONONUCLEAR PHAGOCYTES CULTURED IN VITRO

Uraemic plasma and normal serum were separated into fractions of different molecular sizes by means of molecular filtration through Pellicon filters and liquid chromatography on Sephadex G-15. The amount of substance was considerably higher in uraemic fractions than in comparable normal fractions. When normal human mononuclear phagocytes were cultured in the presence of the uraemic fraction containing the largest molecules, detachment of phagocytes from the glass coverslips was significantly increased. The digestion capacity of the remaining cells was decreased. The results indicate that substances in uraemic plasma responsible for the impaired function of human mononuclear phagocytes cultured in vitro consist of molecules of a molecular weight higher than 10 000.

Mise en évidence et évaluation des “moyennes molécules” de la taille de la vitamine B 12 présentes dans les liquides biologiques de sujets normaux et de patients urémiques

Determination of “middle molecules” presenting vitamin B 12 molecular size in normal and uremic body fluids Uremic solutes with the molecular size of vitamin B 12 are assumed to be toxic. An analytical method is proposed to detect and separate these solutes in body fluids using two combined techniques: gel filtration on Sephadex G-15 and ion-exchange chromatography on DEAE-Sephadex A-25. The vitamin B 12 molecular size has been localized by ultrafiltration through membranes with a defined cut-off. Normal and uremic body fluids (urine, plasma, hemodialysis fluid) have been separated into 9 ultraviolet-absorbing peaks (a to i) by high-speed gel filtration. Peaks b and e present the molecular size of vitamin B 12, 10–15 Å molecular diameter in pH 7 aqueous solution. Peak b, which correlates with uremic neuropathy, is separated into 6 sub-peaks (b 1 to b 6) by ion-exchange chromatography, sub-peak b 4.2 is the only one to correlate with uremic neuropathy. The coefficient of variation in the integrated area of a single peak is 16%. This method gives the chromatographic profile of the vitamin B 12 molecular size content from 500 μl of uremic plasma or 100 μl of normal urine within one hour.

1048 IMMUNE RESPONSE IN UREMIC AND POST-TRANSPLANT CHILDREN

Patients with chronic renal failure and renal allograft recipients have an increased incidence of infection. To define further the defect(s) in immune surveillance, children with chronic uremia, those on maintenance hemodialysis and those with well-functioning renal transplants were studied. Serum IgA, IgG and IgM levels, measured as an indicator of B-cell function, were reduced in all three groups. Intrinsic lymphocyte function was assayed by culturing patient cells in normal pooled plasma and stimulating them with phytohemagglutinin (PHA) or pokeweed mitogen (PWM). Mitogenic activity varied widely within each group but mean values were normal. The presence of an extrinsic inhibitor of lymphocyte activity was examined by culturing normal cells in patient plasma, again stimulating with PHA or PWM. Uremic plasma did not inhibit normal cell response to either PHA or PWM, but both pre- and post-dialysis plasma was markedly inhibitory. Plasma from transplant patients inhibited PHA response but not PWM response. Thus, uremic and transplant patients demonstrate a functional defect in B-cell activity. Although lymphocytes from these patients respond normally to mitogenic stimuli, plasma from dialysis (but not uremic patients) inhibits the mitogenic response of normal lymphocytes. Our data indicate that chronic hemodialysis affects humoral factors which influence lymphocyte activity. Immunosuppressive agents may contribute to the inhibition observed in transplant patient plasma.

Effect of prostaglandin A2 on RNA synthesis in embrionic mouse erythroid cells.

Prostaglandin A2 (PGA2) is shown to stimulate RNA synthesis in 12-day embryonic mouse liver cells, thus expressing an erythropoietin-like effect. This effect was found to be dose-dependent. There was no significant difference between the results observed with patients' plasma before and after dialysis and with or without addition of PGA2. Although the baseline activity of RNA synthesis in the presence of uremic plasma is less than that with normal plasma, the almost equal potentiation of RNA synthesis in erythroid cells incubated with both normal and uremic plasma obtained with PGA2 favors a direct, and not an erythropoietin-mediated, effect of this substance.

Effects of in vivo and in vitro dialysis on plasma transaminase activity.

The effects of dialysis on plasma glutamic oxalacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) were investigated. GOT was measured using Autoanalyzer (SMA) and kinetic (Karmen) methods. Hemodialysis of uremic subjects was associated with a significant increase of GOT (SMA) and GPT (SMA). In contrast, hemodialysis had no effect on GOT (Karmen). However, the SMA method is influenced by substances affecting the blank value. Therefore, the results suggest that the increase in transaminase activities measured by the SMA method are not due to true increases in enzyme activities. Plasma from dialysis patients, obtained prior to hemodialysis, was also dialyzed in vitro. In vitro dialysis of uremic plasma significantly increased GOT (SMA), GOT (Karmen) and GPT vitro dialysis of uremic plasma significantly increased GOT (SMA), OGT (Karmen) and GPT (SMA). The results suggest that an inhibitor of transaminase activity may accumulate in renal failure. In vitro dialysis may remove this inhibitor and thus increase true transaminase activity.

Binding of Hippurate in Normal Plasma and in Uremic Plasma Pre- and Postdialysis

The protein binding of 14C-hippurate has been measured by conventional ultrafiltration techniques in the plasma of normal subjects and in uremic subjects pre- and postdialysis. In addition, the clearance of 14C-hippurate was determined in vitro in both isotonic saline and plasma to assess binding limitations on hippurate removal during dialysis. Binding levels of hippurate in normal subjects of 68+/-1.8% (n = 5) were significantly higher than either postdialysis (48.3+/-15.4%; n = 7) or predialysis (36.6+/-11.7%; n = 7) levels in the same uremic subjects. Actual levels of plasma hippurate were, however, considerably greater in uremics (24.7+/-11.2 mg/dl' n = 7) than in normal subjects (congruent to 0.5 mg%). The difference in hippurate binding between pre- and postdialysis samples in uremics was significantly different from zero (p less than 0.01, t = 5.36), indicating depletion of competitive site-binding species during dialysis. The saline clearance of hippuric acid (99.1 +/-0.5 ml/min; n = 6) under standard conditions in a capillary dialyzer (CDAK-4) was consistent with the expected clearance of a solute of its molecular weight. Hippurate clearance in citrated plasma, where binding was determined as 50+/-3%, was 65+/-0.7 ml/min (n = 6), in good agreement with a theoretically predicted clearance of 60 ml/min for this level of binding. High serum levels of hippurate and its derivatives, may depress effective function of various organs. In addition to the normal dietary intake of hippurate and its precursors, patients on dialysis receive a further burden of hippurate precursor in the form of benzyl alcohol, the common preservative in heparin solutions. The large body burdens of hippurate in dialysis patients, coupled with its impaired removal on dialysis due to binding, point to the necessity for a through investigation of the potential toxicity of this compound.

INHIBITORY EFFECTS OF PLASMA FROM URAEMIC PATIENTS ON HUMAN MONONUCLEAR PHAGOCYTES CULTURED IN VITRO

Human mononuclear phagocytes were cultured in plasma from uraemic patients. The presence of uraemic plasma during the engulfment or digestion of 125I-labelled Candida albicans did not inhibit these functions in mononuclear phagocytes cultured for 8 days under normal condition. When normal human macrophages were cultured in the presence of uraemic plasma for 2-4 days, a marked detachment of the cells from the glass coverslips was registered. The phagocytic function of the remaining cells was impaired. Creatinine, urea and methylguanidine in concentrations higher than those usually measured in plasma from uraemic patients did not influence the functional properties of the cells. The inhibitory effect of uraemic plasma on the mononuclear phagocytes is suggested as an explanation for the increased frequency of infections in uraemic patients.

Modification of Renin Reactivity by Lipids Extracted from Normal, Hypertensive, and Uremic Plasma

Plasma renin reactivity (PRR), the in vitro rate of angiotensin generation after addition of renin, is greater in plasma of hypertensive patients and uremic patients than in plasma of normotensive control subjects. To determine if this difference is due to different substrate reactivities, substrate was denatured and replaced with homologous substrate. After a 180 min incubation, PRR in normal plasma (73 ng/ml +/- 5 SE) was less (P less than 0.01) than that in hypertensive (112 ng/ml +/- 15 SE) or uremic (123 ng/ml +/- 39 SE) plasma. To determine if uremic plasma lacks a renin inhibitor, buffer or plasma was added to renin-renin substrate. Less angiotensin was generated (P less than 0.05) with normal (72 ng/ml +/- 4 SE) and uremic (88 ng/ml +/- 4 SE) plasma during 30 min than with buffer (107 ng/ml +/- 4 SE). After 180 minutes, less angiotensin was generated with normal (P less than 0.05) but not uremic plasma (P greater then 0.6), than with buffer. In vitro angiotensin generation was inhibited by lipids extracted from normal plasma. Lipids were separated into acetone soluble (neutral lipids) and acetone insoluble (phospholipid) fractions. Acetone soluble lipids, extracted from normal plasma, competitively inhibit renin: renin was not inhibited by acetone insoluble lipids. Acetone soluble lipids extracted from uremic plasma inhibited PRR to a lesser extent than lipids from either normal plasma or hypertensive plasma (P less than 0.01). Increased PRR in uremic plasma may be related to the deficiency of a circulating acetone soluble renin inhibiting factor.

Protein binding of salicylate in uremic and normal plasma

Protein binding of salicylate was studied by equilibrium dialysis at 37 degrees C in plasma from uremic patients and healthy subjects. The protein binding was considerably lower in the uremic plasma at all salicylate concentrations studied (14 to 1,400 mug/ml). Scatchard plots of the data were computer-analyzed assuming binding to the classes of binding sites. According to this analysis, the binding to the class of primary binding sites was considerably decreased in the uremic plasma. In addition to the effect of the uremic state, the binding was considerably decreased at high therapeutic plasma levels and at low albumin levels. The combined effect of two or three of these factors may lead to unexpectedly high unbound fractions of salicylate, which should be considered in the monitoring of plasma salicylate levels in patients.

Impaired plasma protein binding of phenytoin in uremia and displacement effect of salicylic acid.

The plasma protein binding of phenytoin (DPH) was studied by equilibrium dialysis at 37 degrees C in plasma from uremic patients and healthy subjects. Scatchard plot analyses demonstrated a decreased association constant Ka for the DPH-albumin interaction in the uremic plasma (mean 1.76 - 10(3) M-1 +/-SD 0.12 and a mean 4.10 - 10(3) M-1 +/- SD 0.24 in normal plasma). Studies on separated fractions of serum did not indicate any significant binding of DPH to proteins other than albumin. The nonlinear mathematical relationship between bound DPH and serum albumin could be linearized at low drug concentrations by plotting the ratio of bound/unbound DPH against albumin concentration. The displacement effect of salicylic acid at a concentration of 276 mug/ml (2mM) and DPH was considerable in plasma from normal subjects. In uremic plasma the effect was of much smaller magnitude.

Inhibition of lipoprotein lipase by uremic plasma, a possible cause of hypertriglyceridemia

In an attempt to define pathogenesis of the previously described impaired triglyceride (TG) removal in uremia, the effects of the addition of normal and uremic plasma on the activity of lipoprotein lipase (LPL) from rat epididymal adipose tissue were examined. Six uremic patients on chronic dialysis and 13 normals were studied. Adding increments of normal and uremic plasma increased the LPL activity to maximal levels when 0.1 ml of plasma was added. Larger aliquots of uremic plasma produced marked inhibition of LPL activity. This inhibition was not observed with the normal plasma. When increasing amounts of uremic plasma were added to an incubation mixture already maximally activated by 0.1 ml of normal plasma, inhibition of LPL was again observed. This inhibition was still present in uremic plasma which had been dialysed against cold saline. The inhibitor was in the lipoprotein-free ( d > 1.225) fraction of the plasma. The results indicate that uremic plasma has an LPL inhibitor which is probably a protein and may play a role in the pathogenesis of uremic hypertriglyceridemia.

Uric acid: Binding levels of urate ions in normal and uraemic plasma, and in human serum albumin

It has recently been suggested that the binding of urate ions to plasma proteins could be: (i) 20 per cent or more in normal subjects and (ii) as high as 10–15 per cent in some uraemic patients. It has also been suggested that oral aspirin administration may enhance urate ion removal on dialysis. These contentions are in conflict with previously published data. In this paper, the binding of urate in plasma is re-examined in vivo, and in vitro, at 37° and at pH 7.4. Conventional ultrafiltration techniques showed that the binding of urate ions is 5 ± 1 per cent in normal human serum albumin (HSA), 10 ± 1 per cent in defatted HSA, 6 ± 1 per cent in normal plasma and 2 ± 1 per cent in the plasma of chronic uraemics. In vivo urate ion binding was estimated on patients undergoing dialysis. These data were in good agreement with the in vitro data. In addition, aspirin administration failed to enhance urate ion clearance on haemodialysis during carefully controlled clinical experiments. Analysis of recent data, suggesting high urate binding levels, indicates that these studies were done under non-physiological conditions. Our data support previous contentions that urate ions are essentially freely filterable at the glomerulus and that binding does not significantly impair urate clearance on dialysis.

Leucocyte sodium transport in uraemia.

1. In sixteen patients with severe chronic renal failure the rate constant for total sodium efflux from leucocytes was significantly reduced compared with that in thirty control subjects. This difference lay chiefly in the glycoside-sensitive ('active') moiety of sodium efflux. 2. In sixteen patients receiving regular haemodialysis, the rate constant for total sodium efflux from the leucocyte was significantly greater than in the undialysed uraemic patients though still subnormal. 3. In individual patients, an increase in sodium efflux could be detected as early as 1 week after regular haemodialysis was started. 4. These results are compatible with the existence of a dialysable molecule in uraemic plasma affecting leucocyte sodium transport.

Failure of methylhistidines to inhibit platelet aggregation at concentrations found in uremic plasma

Methylhistidines are among the amino acids which are present in increased concentrations in the plasma of severely uremic patients who may have a hemorrhagic diathesis. Histidine contains an imidazole ring, and our previous work has shown inhibition of collagen-induced platelet aggregation by imidazole in concentrations as low as 0.5 mM. Collagen-induced, adenosine diphosphate-induced, and norepinephrine-induced platelet aggregation were tested in platelet-rich plasma by a turbidimetric technique after incubation of the plasma with varying concentrations of the methylhistidines for 1 hour. Platelet aggregation was unaffected by methylhistidine concentrations up to 0.6 mM. Only norepinephrine-induced platelet aggregation was slightly inhibited at a concentration of 4.7 (mM far higher than found in uremic patients). The imidazole ring as a portion of the methylhistidine molecule appears to have lost much of its effect on platelet aggregation.

Decreased plasma protein binding of diazoxide in uremia.

The effect of uremia on the binding of diazoxide to plasma proteins was studied. An equilibrium dialysis technique, using diazoxide-minus14C at approximately 30 and 300 mug/ml in the plasma phase, was used to measure diazoxide binding to plasma. Serum albumin concentration (Alb) and serum creatinine (Cr) or blood urea nitrogen (BUN) were negatively correlated. By single regression analysis, per cent free diazoxide (%FD) correlated negatively with Alb and positively with Cr or BUN. When %FD was regressed simultaneously against Alb and Cr or BUN, Alb emerged as the sole determinant of %FD (p less than 0.001), indicating that creatinine or BUN correlated with %FD by their inverse correlation to Alb rather than by an effect on drug protein binding. At the levels of Alb studied, %FD varied over a 2-fold range. In a retrospective study of the influence of uremia on diazoxide effect in hypertensive patients, a relatively low correlation (r, 0.59) was found between BUN and hypotensive effect. Prospective studies involving correlations of drug effect with renal function and %FD are required to assess the clinical importance of decreased binding of diazoxide to uremic plasma.

Evidence for the existence of an acetone soluble renin inhibiting factor in normal human plasma.

After addition of exogenous human renin, the in vitro rate of angiotensin I generation is faster in plasma of patients with chronic renal insufficiency and, to a lesser extent, in plasma of patients with essential hypertension than in plasma from normotensive control subjects. The increased reactivity of renin in hypertensive and uremic plasma is not related to differences of endogenous renin activity, angiotensinase activity, renin substrate concentration, or substrate reactivity. Addition of normal, hypertensive, and uremic plasma to a human renin-sheep renin substrate system inhibited the rate of angiotensin generation, although significantly less inhibition was observed with uremic plasma. The reactivity of renin increased in normal plasma but not in uremic plasma after treatment with 95% acetone. After acetone extraction renin reactivity in normal and plasma inhibited the rate of angiotensin generation in a renin-renin substrate system. Less inhibition occurred with the acetone extract from a pool of uremic plasma. These results provide evidence for the existence of a naturally occurring acetone soluble renin inhibiting factor in normal and uremic plasma. The increased reactivity of renin in uremic plasma may be related to a deficiency of this factor.

Erythrocyte transketolase activity in dialysis patients with neuropathy

Erythrocyte transketolase activity (ETKA) has been studied in a group of uremic patients exhibiting neuropathy on clinical grounds and demonstrating decreased motor nerve conduction velocities in the right peroneal nerve. Single or multiple hemodialyses did not affect ETKA in these patients nor did uremic plasma have any inhibitory effect on ETKA in erythrocytes from nonuremic patients. The enzyme activity actually tended to be greater than normal in uremic patients when related to the erythrocyte count. These results indicate that ETKA is unrelated to the peripheral neuropathy found in uremia.

Uraemic and normal plasma protein binding of various cardiac glycosides under "in vivo" conditions.

Abstract. Plasma from normal subjects and uraemic patients was obtained five minutes after injection of tritiated g‐strophantin, methyldigoxin, digoxin or digitoxin and it was dialyBed (dualclosed loop dialysis; pressure balanced to prevent fluid shift; cuprophane membrane; pH 7.4; 37 °C) against the subject's own plasma‐water obtained by means of haemodiafiltration before injection of the cardiac glycosides.–Plasma protein binding was calculated from the constant concentration difference between plasma and plasma‐water during the diffusion equilibrium after 220 min. of dialysis. This value was significantly reduced in the uraemic patients as compared to the normal subjects, although the plasma protein concentration of the two groups was not different. Methyldigoxin protein binding was reduced from 29.8 to 19.0%, digoxin from 29.4 to 21.3% and digitoxin from 93.7 to 88.3%. Protein binding of g‐strophantin could not be found neither in normal nor in uraemic plasma.–Similar differences between the different cardiac glycosides and the uraemic and normal plasma were found in the mass transfer during the first two hours of dialysis. The conclusion from these results is that in uraemic patients a more rapid onset of action and an enhanced activity of protein‐bound glycosides might be considered.–When interpreting the plasma concentration of cardiac glycosides in uraemic patients it should be remembered that the pharmacological effect depends on the unbound part of the drug, which is increased in uraemia. Although the protein binding of digitoxin is reduced by only 5.4%, the result is a doubling of the unbound glycoside concentration.