Evaluation of blood compatibility of PEO grafted and heparin immobilized polyurethanes.

To develop better blood compatible polymer for long-term biomedical applications, poly(ethylene oxide) (PEO) grafted and/or heparin immobilized polyurethanes (PUs) were made by novel surface modification. Their blood compatibilities were investigated using in vitro platelet adhesion test, APTT, PT, immobilized heparin bioactivity measurement, and ex vivo rabbit A-A shunt test. In platelet adhesion tests, PEO grafted PU surfaces, compared to PU control, displayed very little platelet adhesion and activation, and this effect was more significant as the molecular weight of PEO increased from 200 to 2000. Also, the degree of platelet adhesion was lower in the heparinized PU surfaces than that of PU control. The immobilized heparin showed a greater effect on intrinsic blood coagulation factors than on extrinsic ones, whereas the PU-PEO surface was independent of blood coagulation factors. Lowering both in vitro platelet adhesion and activation led to a prolongation in the ex vivo occlusion time. In particular, the heparinized PU-PEO surfaces displayed enhanced blood compatibility due to the synergistic effects of PEO and heparin.

Intracellular spin-trapping of oxygen-centered radicals generated by human neutrophils.

Phagocytosing neutrophils secrete superoxide into a vacuole generally inaccessible for direct study. However, the spin-trapping agent 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) enters the cytoplasm of several cell types where it can report free radical species including superoxide and hydroxyl radical. In the present study we employed a variety of experimental conditions to eliminate extracellular ESR signals and/or free radicals generated by stimulated neutrophils so that DMPO adducts reported events inside the cell. We identified a concentration of poly(ethylene glycol)-modified superoxide dismutase that permitted measurement of intracellular superoxide as determined by several criteria. It seems likely that poly(ethylene glycol)-modified superoxide dismutase is too large to enter the neutrophil phagosome. Under these conditions no hydroxyl radical was detected, as would be predicted from earlier studies with spin-trapping. Use of poly(ethylene glycol)-modified superoxide dismutase should allow on-line measurement of phagosomal events, thereby improving our understanding of microbicidal and inflammatory processes.

Degradation of plasma bilirubin by a bilirubin oxidase derivative which has a relatively long half-life in the circulation.

To enhance degradation of unconjugated bilirubin in hyperbilirubinemic subjects, we synthesized a bilirubin oxidase (EC 1.3.3.5) (BO) derivative (PEGBO) by covalently linking (2,4-bis[O-methoxy(polyethyleneglycol)]-6-chloro-s-triazine) (PEG) to the enzyme. Intravenously injected BO in rats disappeared from the circulation with a half-life of 2.5 min; the half-life of PEGBO was 190 min. Intravenously injected BO minimally and transiently decreased plasma bilirubin levels in jaundiced Gunn rats and in bile-duct-ligated jaundiced rats. In contrast, PEGBO rapidly and substantially decreased plasma bilirubin levels and the effect persisted for longer than 3 h. Renal dysfunction often occurs in patients with liver diseases. To study the role of bilirubin toxicity for the kidney, functions of transtubular transport for organic anions was measured in bile-duct-ligated jaundiced animals before and after treatment with PEGBO. Bile duct ligation decreased urinary excretion of phenolsulfophthalein (PSP), an organic anion used for renal function test. Treatment of the jaundiced animals with PEGBO increased the rate of PSP disappearance from the circulation and normalized its urinary excretion. Thus, PEGBO might be useful for the study of bilirubin toxicity in jaundiced animals.

Decrease in intestinal permeability to polyethylene glycol 1000 during development in the pig.

Changes in intestinal permeability during postnatal development in the pig were investigated by using different-sized polyethylene glycols in the Mr 766-1338 range (polyethylene glycol 1000) as permeability probes. Pigs of varying age, newborn (Oh), 36-45 h old and 22-28 days old, were gavage fed polyethylene glycol 1000 together with the macromolecular markers bovine serum albumin, ovalbumin or FITC-labelled dextran 70,000. The 4-h blood serum concentrations of the different markers were determined and taken as an estimate of their intestinal transmission. In the newborn pigs, high serum levels of polyethylene glycols were obtained, concomitant with high serum levels of bovine serum albumin and FITC-dextran. After intestinal macromolecular closure in the 36-45 h-old pigs, lower serum polyethylene glycol levels were found, especially of those with a Mr greater than 1100 Da. In the 22-28 days-old pigs, polyethylene glycol levels were reduced to one-tenth or less of those in the 36-45 h-old pigs, with the levels decreasing markedly with increasing molecular size. These results show that there is a correlation between the intestinal permeability of polyethylene glycols, especially those larger than 1100, and macromolecules in the newborn pig around intestinal closure, suggesting that such polyethylene glycols traverse the gut by the macromolecular route. During later development, further intestinal maturation results in a markedly reduced permeability to polyethylene glycol 1000.

Laboratory assessment of poisoning with a carbamate insecticide.

We discuss a case of a 17-year-old white male who intentionally ingested a tick and flea insecticide and was admitted to the emergency room unconscious, with signs and symptoms of cholinergic toxicity. Capillary gas chromatography and electron-impact mass fragmentographic analysis of the patient's urine and serum demonstrated the presence of poly-ethylene glycol and propoxur (o-isopropoxyphenyl N-methyl-carbamate), a carbamate-based cholinesterase inhibitor commonly used in insecticides. The patient fully recovered, but only after a complicated hospital course. We also discuss the laboratory assessment and clinical treatment of poisoning with carbamate and organophosphate insecticides.

Polyethylene glycol-L-asparaginase and L-asparaginase studies in rabbits.

Injections of polyethylene glycol (PEG)-L-asparaginase or L-asparaginase were given to two rabbits each at doses of 40 units/kg. Ten min following injection of either enzyme preparation, the plasma enzyme concentration was approximately 1 unit/ml. This level decreased steadily in the rabbits given L-asparaginase, with a t1/2 of approximately 20 hr. In contrast, the enzyme level in rabbits given PEG-L-asparaginase decreased much more slowly, with a t1/2 of approximately 144 hr. The slower disappearance rate of PEG-L-asparaginase resulted in greater values for the area under the concentration versus time curve and smaller values for total clearance. Immediately following the enzyme injections, no L-asparagine could be detected in the plasma, and a transient elevation of L-aspartic acid levels was noted. By 4 hr, the L-aspartic acid level in all of the rabbits returned to near normal. The L-asparagine, however, was not measurable as long as plasma enzyme was detectable. Levels of L-asparagine returned to normal 4 days after L-asparaginase administration, and 27 days elapsed before L-asparagine was detected in rabbits given PEG-L-asparaginase.

The metabolic sulphation of polyethyleneglycols by isolated perfused rat and guinea-pig livers.

1. It is shown that isolated perfused rat and guinea-pig livers can sulphate polyethyleneglycol 200 at rates of about 5 and 10 nmol/g per min, respectively. 2. In the guinea-pig about equal amounts of sulphated polyethyleneglycol 200 appear in the bile and in the perfusate, while in the rat about 99% appears in the latter. 3. Polyethyleneglycols 400 and 1000 are also sulphated in perfused guinea-pig liver but polyethyleneglycol 6000 is not. 4. Polyethyleneglycols are therefore not suitable solvents for xenobiotics which may undergo sulphation because of the likelihood of competition for sulphate.

Safety evaluation of free radical scavengers PEG-catalase and PEG-superoxide dismutase.

Treatment with catalase and SOD (superoxide dismutase) could diminish the damage due to oxygen free radical formation, but these enzymes are rapidly removed from circulation. The covalent attachment of monomethoxypolyethylene glycol (PEG) to catalase and SOD extended their plasma half-lives. Toxicity of PEG-catalase and PEG-SOD was evaluated in mice and rats prior to their use as free radical scavengers. Rodents used in acute, subacute, and subchronic toxicologic studies could tolerate large doses of PEG-catalase and PEG-SOD without developing toxic signs. The conjugates did not affect survival rate, appearance, behavior, food intake, blood chemistry, hematology, or urinalysis. In general, body weight gains, organ weights, and histomorphology were also unaffected. Massive doses of PEG-catalase caused slight weight loss, splenic hypertrophy, and generalized splenic stimulation in mice. Massive doses of PEG-SOD resulted in vacuolation in splenic macrophages in rats. PEG-catalase and PEG-SOD circulated for 3 days and 8 days, respectively, in mice following i.v. or i.m. administration.

Disposition of intravenous glycofurol: effect of hepatic cirrhosis.

The disposition of intravenous glycofurol, a solvent for some drugs, was followed in nine male patients with hepatic cirrhosis. In comparison with age-matched controls, glycofurol clearance in the patients with cirrhosis was reduced 43% and terminal t 1/2 was prolonged 103%. There was no significant difference between the cirrhotic and control groups in volume of distribution.

Appearance of 14C-polyethylene glycol 4000 in intestinal venous blood: influence of osmolarity and laxatives, effect on net water flux determination.

In anaesthetized rats the appearance rate of 14C-polyethylene glycol 4000 (14C-PEG 4000) was measured in the intestinal venous blood after intraluminal administration into a jejunal, ileal, and colonic segment. The absorption rate was small, especially in the colon (0.5-4.2% within 60 min in an intestinal segment of about 300 mg wet tissue weight depending on the batch of 14C-PEG 4000). The absorbed PEG in the plasma consisted mainly of molecules with lower molecular weight than 4000 which were included in the commercial batches of 14C-PEG 4000. The appearance rate of 14C-PEG decreased with time after single dose but remained constant, when a 14C-PEG solution was perfused continuously through the intestinal lumen. A hypotonic solution increased and a hypertonic one decreased slightly the absorption of PEG in the jejunum and ileum but not in the colon. The influence of bisacodyl (100 mgl(-1)) and ricinoleate (10 mmol l(-1)) on the absorption of PEG was small or absent, while deoxycholate (5 mmol 1(-1)) raised the absorption rate considerably, predominantly of the high molecular weight fraction. If in intestinal absorption studies a batch of commercial 14C-PEG 4000 with a small low molecular weight fraction is used, the error in the determination of net water flux caused by the absorption of PEG can be neglected. The influence of osmolarity and laxatives is insignificant. Bile acids increase the intestinal permeability of PEG 4000, so that the net water flux determination can be biased considerably.

Polyethylene glycol intoxication in burn patients.

Unexplained increases in the anion gaps and serum osmolalities were observed in 3 burn patients who died following treatment with a polyethylene glycol-based burn cream. Toxicity due to absorption of polyethylene glycol was suspected after other causes had been excluded. Ethylene glycol was found in the circulation and the 3 patients died in acute renal failure. All the patients were acidotic with increased anion and osmolal gaps. This syndrome was similar to the more common poisoning with ethylene glycol but also included an increased serum calcium with a concomitant decrease in the ionized calcium. The cause of this high 'calcium gap' appeared to be binding of calcium by dicarboxylic acid metabolites of polyethylene glycol. These findings were the same as those found in rabbits treated with polyethylene glycol.

Alteration of the circulating life and antigenic properties of bovine adenosine deaminase in mice by attachment of polyethylene glycol.

Polyethylene glycol was attached covalently to adenosine deaminase (ADA) using cyanuric chloride as the coupling agent. The modified adenosine deaminase (PEG-ADA) appears to lose its immunogenicity in mice following multiple intravenous injections. PEG-ADA does not react with antibodies raised against native ADA. The circulating half-life (T1/2) of PEG-ADA was increased to 28 hr. The lack of detectable antibody formation and long circulating life may make PEG-ADA suitable for treating human ADA deficiency.

Preparation of a polyethylene glycol: superoxide dismutase adduct, and an examination of its blood circulation life and anti-inflammatory activity.

Methoxypolyethylene glycol (PEG) of 5000 daltons was attached covalently to 19 of the 20 available lysine residues of bovine erythrocyte superoxide dismutase. The adduct (PEG-superoxide dismutase) has 51% of the enzymatic activity of superoxide distmutase. PEG-superoxide dismutase exhibits a sharply enhanced serum circulating life during repetitive intravenous injections compared to the native enzyme. While no evidence of an immune response to repetitive injections of PEG-superoxide dismutase is observed, the unmodified enzyme appears to produce slight sensitization in the form of quicker removal from the serum after 13 injections over a period of 30 days. Antisera to superoxide dismutase and PEG-superoxide dismutase, however, produce no detectable antibodies as determined by the Ouchterlony method. Intraperitoneally injected PEG-superoxide dismutase enters the bloodstream more readily than superoxide dismutase. PEG modification slightly improves the enzyme's anti-inflammatory activity, which was observed in rats over a period of eight days.

Detection of immune complexes on the surface of polymorphonuclear neutrophils.

A simple immunohistological test has been developed to detect and to quantitate the presence of immune complexes (IC) on the surface of human polymorphonuclear neutrophils (PMN). The interaction between IC and PMN has been evaluated in several human IC diseases. High amounts of immunoglobulins (Ig) and C3 were detected on the PMN surface from these patients. The deposits of Ig and C3 were inversely related to the percentage of membrane-free receptors for Fc and C3.

Ethylene and diethylene glycol toxicity.

1. Blood concentrations of ethylene and diethylene glycol were evaluated in Sprague-Dawley rats at varying intervals following oral dosages of the glycols. 2. Ethylene and diethylene glycol in rat blood stored under refrigeration at 4 degrees +/- 10 degrees C for a period of 30 days exhibited minimal concentration losses, contrary to previous reports. 3. The amount of oxalate in the blood and kidneys of Sprague-Dawley rats doses with ethylene and diethylene glycol was quantitated. The animals dosed with ethylene glycol demonstrated significantly higher oxalate levels, particularly at 8 hr post-dosing, than similar animals dosed with diethylene glycol. 4. Ethylene glycol induced oxalate deposition within the kidney without significant histologic changes. Diethylene glycol induced histologic changes within the kidneys without kidney oxalate deposition. 5. Maximal kidney oxalate levels, following ethylene glycol dosage, occurred concurrently with peak blood oxalate concentrations. In the case of diethylene glycol, kidney oxalate levels did not peak until 4 hr after maximal blood oxalate levels. 6. Ethylene and diethylene glycol induced different modes of death in Sprague-Dawley rats.

The influence of Cortisone on the hepatic uptake of Triton WR-1339 in adrenalectomized rats.

The effect of cortisone acetate on the hepatic uptake of Triton WR-1339 was studied in adrenalectomized rats. The hormone was found to retard the uptake of Triton in the liver, and at the same time reduce the plasma clearance of this compound. The inhibitory effect of the hormone on endocytosis was seen in purified preparations of both Kupffer cells and parenchymal cells. Triton renders the liver lysosomes progressively lighter. The change in equilibrium density (in sucrose gradients) was found to occur more rapidly in hormone-treated animals. The possibility that cortisone reduces the number and increases the size of the lysosomes is discussed. Our data indicate that the uptake of Triton in non-parenchymal (Kupffer) cells represents about 20% of the total hepatic uptake.