Exchange of macromolecules between plasma and peritoneal cavity in ascites tumor-bearing, normal, and serotonin-injected mice.

Fluorescein-labeled dextrans (FITC-D) from 3 to 5000 kDa (Stokes' radii from 1 to 40 nm) were used to study influx from the plasma into the peritoneum and efflux from the peritoneal cavity into the plasma in normal and ascites tumor-bearing mice and in mice whose peritoneal vessels had been rendered hyperpermeable by serotonin. Two syngeneic transplantable murine ascites tumors were studied: mouse ovarian tumor and the TA3/St breast adenocarcinoma. To control for effects of peritoneal fluid volume, influx and efflux were also analyzed in mice that had received 5 ml of 5% bovine serum albumin i.p. as "artificial ascites." Following i.v. or i.p. injection, levels of FITC-D in the plasma and peritoneal fluid were quantitated by fluorimetry at successive time intervals from 5 to 360 min posttracer injection. Influx and efflux data were analyzed with a model consisting of three compartments (plasma, peritoneal cavity, and the extravascular space of all other organs) to yield kinetic parameters that characterized macromolecular transport. Depending on the size of the FITC-D tracer, from 3- to 50-fold more FITC-D accumulated in mouse ovarian tumor or TA3/St tumor ascites fluid, and 3- to 10-fold more FITC-D accumulated in the peritoneum of serotonin-treated than normal mice, all of it intact by gel exclusion chromatography. Influx of the FITC-D from plasma into the peritoneum, as characterized by the rate constant k1, was 2- to 40-fold greater in ascites tumor-bearing animals and 2- to 10-fold greater in serotonin-treated animals than in controls. Control animals with artificial ascites showed at most a 4-fold increase in the value of k1. As judged by fluorescence microscopy, the permeability of peritoneal-lining vessels in ascites tumor-bearing animals was greatly increased to FITC-D of 70 to 5000 kDa. Efflux of FITC-D, characterized by the rate constant k2, was reduced from 5- to 50-fold in ascites tumor-bearing animals but was unchanged or actually somewhat enhanced following serotonin treatment. Efflux in animals that had received artificial ascites was reduced 2.5- to 12.5-fold, correlating increased peritoneal fluid volume with decreased efflux. We conclude that tracer accumulation in malignant ascites fluid results from both increased influx as well as impaired efflux. Influx, and to a lesser extent efflux, were significantly affected by tracer size. However, within the range of FITC-D tested, we found no absolute size barrier to macromolecular transport from plasma to the peritoneal cavity, or vice versa.(ABSTRACT TRUNCATED AT 400 WORDS)

Macrophage function in murine allogeneic bone marrow radiation chimeras in the early phase after transplantation.

We tested several of the functions of macrophages (M phi) in the early phase after allogeneic bone marrow transfer to get information about this important aspect of the nonspecific immune system in the T-cell-deficient recipient. On days 3-5 after transfer, the number of M phi was reduced in the spleen, liver, lungs, and peritoneal cavity (Pe). The phagocytosis of sheep red blood cells (SRBC) by these M phi was normal or even enhanced, as in the case of Pe-M phi. Already on days 8-12 after transfer, the number of M phi in spleen and liver exceeded that of controls, whereas the number was still reduced in lungs and Pe. We examined their ability to kill P815 tumor cells, to produce tumor necrosis factor-alpha (TNF alpha), to phagocytose SRBC, to produce reactive oxygen intermediates (ROI) in vitro and to kill Listeria monocytogenes in vivo. Most functions were normal and often even enhanced, depending on the organ origin, but the ability of Pe-M phi to produce ROI was reduced. Proliferative response to macrophage colony-stimulating factor (M-CSF) and killing of YAC-1 tumor cells revealed a high frequency of macrophage precursor cells in the spleen and liver and a high natural killer (NK) activity in the liver. Altogether, enhanced nonspecific immune function, especially preactivated M phi, may enable chimeras to survive attacks by opportunistic pathogens.

Superoxide production of peritoneal macrophages in experimental gram-negative sepsis; influence of in vitro and in vivo supplements of zinc.

Although zinc is essential for the optimum function of the immune system, there is some controversy regarding treatment with zinc during acute infections where low serum zinc levels are often recorded. The aim of the present study was to investigate the influence of in vitro and in vivo zinc supplementation on the potentially toxic metabolic activity of peritoneal macrophages during infection. Rats were made septic by implanting a gelatin capsule containing known amounts of E. coli, and Bacteroides fragilis into the abdomen. Peritoneal macrophages were harvested by peritoneal lavage 72 hours after the induction of sepsis. Superoxide release was measured after stimulation with phorbol myristate acetate (PMA) or serum treated zymosan (STZ). Macrophages from septic rats released significantly higher amounts of superoxide compared with macrophages from sham operated controls after stimulation with both PMA and STZ. Following in vitro supplementation, zinc inhibited the superoxide production of macrophages harvested from septic rats after stimulation with both PMA and STZ. In vivo supplementation with zinc resulted in increased superoxide production from septic macrophages when stimulated with STZ, whereas stimulation with PMA produced no significant changes. Thus, in vitro incubation inhibited the superoxide production of peritoneal macrophages in intraabdominal sepsis, whilst in vivo administration of zinc produced no such effect, and the effect seemed to vary depending on the stimuli used to initiate the respiratory burst.

Utilization of a murine model to optimize volume and dwell time of intraperitoneal cisplatin.

The pharmacokinetics of intraperitoneal (ip) cisplatin were investigated in a murine model. Groups of six animals were studied at initial cisplatin concentrations (Ci) of 50, 100, 200, and 400 micrograms/ml, and at injection volumes (V) of 0.5, 1, 2, and 4 ml. For each Ci and V, four dwell times (Dt) between 1.5 and 120 min were studied. At 180 min mice were killed and five tissues (heart, kidney, liver, bowel, and peritoneum) were obtained for platinum measurement. Platinum recovered from the peritoneal cavity at the end of the dwell time and platinum tissue levels were determined by a radioactive tracer assay using Pt 195m-labeled cisplatin. A total of 384 mice were studied. The permeability times the effective surface area product (pS) was found to be independent of Ci, V, and Dt. Platinum tissue levels were proportional to absorbed dose. As the ratio of platinum tissue level to absorbed dose is constant, it is not possible to change Ci, V, nor Dt to minimize toxicity from a given absorbed dose. This study suggests that the therapeutic index of an individual intraperitoneal cisplatin treatment will not be changed by modifying Ci, V, nor Dt.

Release of eicosanoids in rat peritoneal cavity stimulated with platelet-activating factor (PAF). Effect of the PAF-antagonist BN-52021.

Platelet activating factor (PAF; 10 micrograms) was injected in the peritoneal cavity of rats in the absence or presence of the PAF antagonist BN-52021 (5 mg/kg). Thirty min later, the peritoneal cavity was washed with 3 ml of saline, the fluid was collected and the concentrations of selected eicosanoids were measured using novel enzyme immunoassays. PAF increased by 2.9, 2.8 and 1.7 fold the levels of thromboxane B2, prostaglandin E2 and leukotriene B4 respectively in the peritoneal fluid. The stimulatory effects of PAF was reduced by 42, 51, and 86% for thromboxane B2, prostaglandin E2 and leukotriene B4 respectively by the specific PAF antagonist. These results confirm the presence of specific PAF receptors in tissues and/or cells of rat peritoneal cavity and underline the complex interactions between PAF and eicosanoids.

Determination of peritoneal glucose kinetics in rats: implications for the peritoneal implantation of closed-loop insulin delivery systems.

Peritoneal glucose kinetics were evaluated in the anaesthetized rat, to assess whether the peritoneal cavity would be a suitable site for the implantation of membrane-protected islets of Langerhans (bioartificial pancreas) or the glucose sensor of an artificial B cell. Glucose was measured in peritoneal fluid samples aspirated by needle puncture. Basal peritoneal and blood glucose concentrations were identical in 16 h fasted (n = 4) and non fasted (n = 3) animals. After 10 min of an i.v. glucose infusion (n = 15) the increment in peritoneal glucose concentration was 63 +/- 3% of the increment in blood glucose concentration and both values were significantly correlated (r = 0.92; p less than 0.001). After 10 min of glucose clamping (12.6 +/- 0.8 mmol/l), the increment in peritoneal glucose concentration was 69 +/- 3% (n = 5; p less than 0.05) of the increment in blood glucose concentration. In three additional experiments it was 93 +/- 3% of the increment in blood glucose concentration (NS), after 30 min of glucose clamping (8.0 +/- 0.5 mmol/l). Peritoneal glucose concentration monitored by a glucose sensor: (a) followed blood glucose sluggishly during a glucose clamp (n = 5), confirming the data shown above, (b) followed blood glucose with a 5 min delay and reached the same plateau after the intravenous injection of 1U insulin (n = 3; NS). We conclude that peritoneal glucose reflects blood glucose at basal state and during variations of glycaemia, nevertheless, presenting heterogeneous kinetics. These kinetics might be appropriate for a bioartificial pancreas but not for an in vivo calibration procedure, of a peritoneally implanted glucose sensor.

Body fluid and endometrial concentrations of ketoconazole in mares after intravenous injection or repeated gavage.

After single oral administration of ketoconazole (30 mg/kg bodyweight [bwt]) in 50 ml of corn syrup to a healthy mare, the drug was not detected in serum. Ketoconazole in 0.2 N HC1 was administered intragastrically to six healthy adult horses in five consecutive doses of 30 mg/kg bwt at 12 h intervals. Ketoconazole concentrations were measured in serum, synovial fluid, peritoneal fluid, cerebrospinal fluid (CSF), urine and endometrium. Mean peak serum ketoconazole concentration was 3.76 micrograms/ml at 1.5 to 2 h after intragastric administration. Mean peak synovial concentration was 0.87 micrograms/ml 3 h after the fifth dose. Similarly, mean peritoneal concentration peaked 3 h after the fifth dose at 1.62 micrograms/ml. Mean endometrial concentrations peaked at 2.73 micrograms/ml 2 h after the fifth dose. Ketoconazole was detected in the CSF of only one of the six mares at a concentration of 0.28 micrograms/ml 3 h after the fifth dose. The highest measured concentration of ketoconazole in urine was 6.15 micrograms/ml 2 h after the fifth dose. A single intravenous injection of ketoconazole (10 mg/kg bwt) was given to one of the six mares; the overall elimination rate constant was estimated at 0.22/h and bioavailability after oral administration was 23 per cent.

Simulations of peritoneal solute transport during CAPD. Application of two-pore formalism.

Blood peritoneal clearances of various endogenous solutes in patients undergoing continuous ambulatory peritoneal dialysis (CAPD) were evaluated according to recent developments of the two-pore theory of membrane permeability, using a non-linear transport formalism for the analysis. Based on results obtained from these calculations and taking lymphatic drainage into account, transport from peritoneal cavity to the blood was also simulated. With respect to solute transport the data were compatible with a functional blood-peritoneal barrier consisting of a two-pore membrane containing a large number of paracellular "small pores" of radius 40 to 55 A and a small number of "large pores" of radius 200 to 300 A. Solutes smaller than 25 A in radius were found to be permeating across the peritoneal membrane mainly by means of diffusion across the small pores, whereas solutes larger than 40 A were calculated to reach the peritoneal cavity exclusively by unidirectional convection across the large pores. In addition, water was simulated to be transported through transcellular "ultrapores" (radius less than 8 A) not accessible to hydrophilic solute permeation. Small solute absorption from the peritoneal cavity was found to occur by diffusion across small pores. Molecules larger than 25 to 30 A in radius (molecular weight above 25,000) were simulated to be absorbed from the peritoneal cavity exclusively via non-size-selective lymphatic drainage.

Absorption of plasma proteins from peritoneal cavity of normal rats.

The present study was undertaken to examine whether the uptake of plasma proteins from the peritoneal cavity is quantitative so that tracers could be introduced that way for measuring their turnover. To this end, the metabolic behavior of seven homologous plasma proteins, labeled with 125I, was compared in rats after intravenous or intraperitoneal administration. The animals were maintained under physiological conditions. Total body radiation measurements showed that the degradation rates of albumin, immunoglobulins A and G, alpha 1-macroglobulin, and transferrin were the same regardless of the route of injection. This implies that these proteins are quantitatively absorbed from the peritoneum without undergoing modifications. The half-life of intraperitoneally injected alpha 1-acid glycoprotein was consistently shorter by an average 9%, thus suggesting that this protein becomes slightly altered if introduced that way. Only one-half of intraperitoneally injected fibrinogen survived normally, whereas the other underwent rapid degradation. The surviving molecules had the same half-life as fibrinogen injected intravenously. The fraction of surviving fibrinogen could be augmented by mixing the dose with serum. Within a wide range of concentrations and quantities injected, the degradation rate of transferrin remained the same. Analysis by deconvolution of the plasma curves of albumin and alpha 1-macroglobulin absorbed from the peritoneum showed that the transport process was independent of protein size and, at least up to 35 mg, of the amount injected. According to the same technique, intraperitoneally administered diferric transferrin retained its iron during passage into the circulation.

Free radical generation during primary infections with Nippostrongylus brasiliensis.

The production of free radicals during infection of the rat with Nippostrongylus brasiliensis was investigated. Lipid peroxidation, which is the best documented effect of free radicals, was monitored in the small intestines of infected rats by measurement of malonyldialdehyde and was found to be increased at the time of worm rejection. The capacity of peritoneal leucocytes to produce free radicals, as measured by chemiluminescence, was monitored in rats infected with different doses of N. brasiliensis. Rejection of N. brasiliensis from rats infected with 6000 third-stage larvae (L3) began 2 days earlier than in rats infected with only 600 L3. Maximal free radical generation also occurred 2 days earlier and was quantitatively greater in rats infected with 6000 L3. Free radical generation by leucocytes in response to live adult N. brasiliensis was enhanced by plasma from infected rats indicating the existence of a plasma-borne factor responsible for the initiation of free radical generation in response to N. brasiliensis.

The effect of antioxidants on the rejection of Nippostrongylus brasiliensis.

The effect of the antioxidants, butylated hydroxy anisole (BHA) and vitamin E, on the rejection of Nippostrongylus brasiliensis from the small intestine of the rat was investigated. Worm expulsion was inhibited by BHA. Malonyldialdehyde production in the small intestines and free radical generation by peritoneal leucocytes from infected rats were also inhibited by BHA. Vitamin E, although inhibiting malonyldialdehyde production, did not prevent worm expulsion. Significantly, vitamin E was much less effective than BHA at reducing free radical generation by rat leucocytes in response to N. brasiliensis.

Studies on the accumulation of serum proteins in zymosan-induced inflammation in mouse peritoneum.

Inflammatory responses were induced in mice by intraperitoneal (i.p.) injection of zymosan. This resulted in a rapid accumulation of protein in the peritoneum that was dependent on the time of the injection and concentration of zymosan used. Though other stimuli, e.g., phorbol myristate acetate, lipopolysaccharide, carrageenan and latex beads, caused the accumulation of proteins, the maximum response was obtained only with zymosan. Injection of free fatty acids were unable to induce protein accumulation in peritoneum. Factors which decreased leukotriene production in mouse peritoneum, i.e., dietary n-3 fatty acids essential fatty acid deficient diets, did not affect protein accumulation. Direct injection of leukotrienes also failed to induce protein accumulation. Analyses revealed that the proteins were similar to serum proteins, indicating that zymosan causes the leakage of serum proteins into peritoneum.

Modulation of proline and glucosamine incorporation into tissue repair cells by peritoneal macrophages.

The purpose of this study was to determine the patterns of [14C]proline and [14C]glucosamine incorporation by tissue repair cells (TRC) as modulated by postsurgical macrophages. Rabbits underwent a midline laparotomy followed by resection (2.0 cm) and reanastomosis of their ileum. Another group of rabbits underwent peritoneal wall abrasion with sterile gauze until punctate bleeding developed. Postoperative (1-28 days) exudate cells (PEC) were recovered from the peritoneal cavity after reanastomosis, and (TRC) were obtained directly from the injured peritoneal surface after abrasion. Since the postsurgical exudate was composed mainly of macrophages, we examined the effect of postsurgical macrophage-spent media on the incorporation of [14C]proline, [14C]glucosamine, and [3H]thymidine by TRC. After 7 days of culture, Postsurgical Day 7 TRC were incubated with spent media from postsurgical PEC (greater than 90% macrophages). When TRC were cultured with macrophage-spent media, the number of TRC increased significantly compared to that of fresh medium-treated controls. The incorporation of [3H]thymidine by TRC was also enhanced by macrophage-spent media. The incorporation of [14C]proline and [14C]glucosamine by TRC was also enhanced when incubated with macrophage-spent medium. However, when data were expressed on a per cell basis, incorporation of [14C]proline and [14C]glucosamine by TRC cultured with macrophage-spent media was the same or less than that by cells incubated with fresh medium. These data suggest that the increase in incorporation of glucosamine and proline into connective tissue protein by postsurgical repair cells may be directly modulated by macrophages recruited in response to surgical injury and that this increase is due to the fibroproliferative effect of postsurgical macrophages.

Release of eicosanoids in rat peritoneal cavity during the Arthus reaction. Effect of the PAF-antagonist BN-52021 and indomethacin.

An Arthus reaction was induced in the rat peritoneal cavity. The inflammatory exudates were collected 10 min after induction of the reaction and analysed for the presence of prostaglandin E2 (PGE2) and thromboxane B2 (TXB2) by enzyme immunoassays (EIA), and of leukotriene B4 (LTB4) by radioimmunoassay. Our results showed that control release (CONT) of eicosanoids in the peritoneal cavity averaged 2.3 ng/ml for TXB2, 0.21 ng/ml for PGE2 and 18 pg/ml for LTB4. Following antigen challenge, the levels of TXB2, PGE2 and LTB4 in the peritoneal cavity increased to 17.0 ng/ml, 0.41 ng/ml and 49.0 pg/ml, respectively. Indomethacin totally inhibited the release of PGE2 and TXB2 whereas it increased by 326% the release of LTB4. The PAF antagonist, BN-52021 significantly inhibited (around 40%) the release of LTB4 in rat peritoneal cavity, increased the release of PGE2, and did not affect the release of TXB2. These results clearly suggest a mediatory role for both cyclooxygenase and lipoxygenase products in Arthus reaction and provide evidence that PAF is also involved in complex interactions with the eicosanoids.

The pharmacokinetics of ceftriaxone and cefotaxime in cirrhotic patients with ascites.

We have compared in two separate studies the kinetics of ceftriaxone and cefotaxime in 8 cirrhotic patients with ascites and 8 control subjects after a single 20 min intravenous infusion of 1 g of each drug. The apparent volumes of distribution (Vz) were found to be significantly higher in cirrhotics than in control subjects (0.87, versus 0.49, l.kg-1, for cefotaxime and 0.23 versus 0.13 for ceftriaxone). The elimination kinetics of ceftriaxone were similar in the two groups. In contrast, the total and non-renal clearances of cefotaxime were reduced in cirrhotic patients. The two drugs rapidly entered the ascitic fluid. Peritoneal concentrations of ceftriaxone were higher than 7 micrograms.ml-1 from the second hour after the infusion and were 8.9 micrograms.ml-1 at 24 h. Peritoneal concentrations of cefotaxime were higher than 4 micrograms.ml-1 from 0.5 to 8 h after the infusion.

Preferential hydrolysis of oxidized phospholipids by peritoneal fluid of rats treated with casein.

1-Palmitoyl-2-azelaoyl-PC, which is one of the possible cytotoxic products generated by the oxyhemoglobin-induced lipid peroxidation of 1-palmitoyl-2-linoleoyl-PC, was found to be efficiently hydrolyzed by the peritoneal fluid of rats treated with casein. The rate of hydrolysis of 1-palmitoyl-2-azelaoyl-PC was approx. 15-fold higher than that observed with 1-palmitoyl-2-linoleoyl-PC. When 1-palmitoyl-2-linoleoyl-PC pretreated with oxyhemoglobin was incubated with the peritoneal fluid, oxidized products of PC were hydrolyzed more efficiently than the intact 1-palmitoyl-2-linoleoyl-PC. When 1-[(1-)14C]palmitoyl-2-azelaoyl-PC was incubated with the peritoneal fluid, radiolabeled lysoPC was formed, whereas radiolabeled neutral lipids were not formed, indicating that the hydrolytic activity was of the 'phospholipase A2' type. We previously found and purified an extracellular phospholipase A2 (Chang, H.W. et al. (1987) J. Biochem. 102, 147-154) in the peritoneal fluid of rats injected intraperitoneally with casein. Hydrolysis of 1-palmitoyl-2-azelaoyl-PC by this purified phospholipase A2 was as low as that of 1-palmitoyl-2-linoleoyl-PC. These two phospholipase A2 activities showed different pH optima and Ca2+ requirements. The present phospholipase A2 activity, which preferentially hydrolyzes oxidized products of PC, may play an important role in detoxification or repair of damaged membrane in inflamed sites.

Transperitoneal movement and pharmacokinetics of cefotiam and cefsulodin in patients on continuous ambulatory peritoneal dialysis.

The kinetics of cefotiam and cefsulodin were studied in plasma and dialysate after intravenous and intraperitoneal administration of 1 g to patients undergoing continuous ambulatory peritoneal dialysis. Instillation of autologous hemoglobin as a marker permitted calculation of the cavity volume and, hence, the rate of transfer to and from the peritoneal cavity with time. The patients were divided into 4 groups. Groups 1 and 2 were intravenously given cefotiam (5 patients) and cefsulodin (4 patients), respectively. Groups 3 and 4 (5 patients each) were given cefsulodin intraperitoneally. Group 3 did not have peritonitis, while the patients in Group 4 were studied during peritonitis. Blood and dialysate samples were obtained at selected times during the 5-hour dwell and, for plasma, until 24 hours after drug administration. Pharmacokinetic analysis of the data showed that only 6.0 and 8.7% of the intravenous doses of cefotiam and cefsulodin, respectively, were recovered in the dialysate at the end of the dwell. The net amounts of cefsulodin lost from the dialysate after intraperitoneal administration were 81 and 84%, in Groups 3 and 4 respectively. The peritoneal transfer clearances (using a unidirectional clearance model), calculated after intravenous (17 +/- 10 ml/min, Group 2) and intraperitoneal (17 +/- 5 ml/min, Group 3) administrations were the same. Mass balance of cefsulodin in the body and in the dialysate after intraperitoneal administration indicated that a significant amount (40%, Group 3) of the dose is unaccounted for. One explanation for this imbalance is retention of the drug in the peritoneal lining. This hypothesis is supported by the retention being lower in the peritonitis patients (less than 20%, Group 4), for whom the linings are expected to be partially eroded.