Effect of PD fluid instillation on the peritonitis-induced influx and bacterial clearing capacity of peritoneal cells.

BACKGROUND: The commonly used peritoneal dialysis fluids contain glucose as the osmotic agent. Heat sterilization leads to the formation of glucose degradation products which contribute, together with glucose, to the formation of advanced glycation end-products (AGEs). AGEs have been shown to be present in the peritoneal cavity. Methods have been developed to minimize the amount of glucose degradation products in peritoneal dialysis fluids. In a rat peritoneal dialysis model, we compare the effect of a commonly used peritoneal dialysis fluid, Gambrosol, with a newly developed peritoneal dialysis fluid, PD-Bio, on the influx and functional capacity of the peritoneal cells after 2 weeks of peritoneal dialysis fluid instillation. METHODS: Three groups of animals were used: rats received daily infusion with 15 ml of either 4% Gambrosol (group 1) or 4% PD-Bio (group 2), and a control group of animals did not receive fluid (group 3). After 2 weeks of PD fluid instillation, all the animals were injected with a 0.5 ml suspension containing 3x10(8) colony-forming units of Staphylococcus aureus. The in vivo bacterial clearing capacity was determined after 15 h. RESULTS: A statistically significant higher leukocyte influx was found in the control group compared with both PD fluid-injected groups. No statistical differences in bacterial clearing were observed among the three groups, although the number of bacteria recovered from the PD-Bio group tended to be lower than that from the Gambrosol group. Moreover, in both PD fluid instillation groups, the bacteria tended to be cleared more slowly compared with the control group. The number of mesothelial cells in the PD fluid groups was significantly greater than in the control group. CONCLUSION: No differences were observed in bacterial clearing capacity, leukocyte influx and mesothelial cell number after a 2 week exposure of the peritoneal cavity to Gambrosol vs PD-Bio.

Are aldehydes in heat-sterilized peritoneal dialysis fluids toxic in vitro?

OBJECTIVE: Chemical analysis of several brands of peritoneal dialysis fluids (PD fluids) has revealed the presence of 2-furaldehyde, 5-HMF (5-hydroxymethylfuraldehyde), acetaldehyde, formaldehyde, glyoxal, and methylglyoxal. The aim of this study was to investigate if the in vitro side effects caused by glucose degradation products, mainly formed during heat sterilization, are due to any of these recently identified aldehydes. DESIGN: Cell growth media or sterile filtered PD fluids were spiked with different concentrations of thealdehydes. MEASUREMENTS: In vitro side effects were determined as the inhibition of cell growth of cultured mouse fibroblasts or stimulated superoxide radical release from human peritoneal cells. RESULTS: Our results demonstrate that the occurrences of 2-furaldehyde, 5-HMF, acetaldehyde, formaldehyde, glyoxal, or methylglyoxal in heat-sterilized PD fluids are probably not the direct cause of in vitro side effects. In order to induce the same magnitude of cell growth inhibition as the heat-sterilized PD fluids, the concentrations of 2-furaldehyde, glyoxal, and 5-HMF had to be 50 to 350 times higher than those quantified in the PD fluids. The concentrations of acetaldehyde, formaldehyde, and methylglyoxal observed in the heat-sterilized PD fluids were closer to the cytotoxic concentrations although still 3 to 7 times lower. CONCLUSION: Since none of these aldehydes caused in vitro toxicity at the tested concentrations, the toxicity found in PD fluids is likely to be due to another glucose degradation product, not yet identified. However, it is possible that these aldehydes may still have adverse effects for patients on peritoneal dialysis.

In vitro biocompatibility of a heat-sterilized, low-toxic, and less acidic fluid for peritoneal dialysis.

OBJECTIVE: The aim of this study was to investigate a peritoneal dialysis (PD) fluid (PD-Bio), produced with the intention of reducing the amount of glucose degradation products and to increase the final pH. The heat sterilization of the fluid was performed with the glucose separated from the electrolytes. After sterilization the two solutions were combined. METHODS: The in vitro biocompatibility of PD-Bio was measured as the inhibition of cell growth of a cultured fibroblast cell line and as the stimulated release of interleukin-1 beta from cultured human mononuclear cells. The glucose degradation products were measured as UV absorbance at 228 nm or 284 nm and the concentration of aldehydes was estimated with high-performance liquid chromatography and gas chromatography. RESULTS: Our results demonstrate that in comparison to conventional PD fluids the pH of PD-Bio was increased, to about 6.5. Due to less contaminating glucose degradation products in PD-Bio, basal cytotoxicity was significantly decreased for both 1.5% and 4% glucose-containing fluids, and the stimulated release of interleukin-1 beta was normalized compared to sterile filtered controls with the same pH. UV absorbance measured at 228 nm was decreased, whereas the absorbance at 284 nm was equal to that of a conventional fluid. In PD-Bio the concentrations of formaldehyde, acetaldehyde, methylglyoxal, and 2-furaldehyde were found to be below the detection limit, whereas glyoxal was present in the same and 5-hydroxymethylfurfural (5-HMF) in higher concentrations than in conventionally produced PD fluid. CONCLUSIONS: The results demonstrate that it is possible to improve biocompatibility of PD fluids by simply changing the way the fluid is produced.

Heat sterilized PD-fluids impair growth and inflammatory responses of cultured cell lines and human leukocytes.

We have recently demonstrated that commercial PD-fluids inhibit the growth of a cultured mouse fibroblast cell line. Toxic substances produced during heat sterilization were believed to be the probable cause of the growth inhibition. The aim of the present study was to investigate if heat sterilized PD-fluids affect other cell types and other cellular functions than the growth of fibroblasts. The effect of three commercially and one laboratory made PD-fluid on cell growth of a mouse macrophage cell line (RAW) and a human neuroblastoma cell line (SH-SY5Y) was examined. The influence on stimulated release of tumour necrosis factor alpha (TNF alpha) from the macrophage cell line and stimulated superoxide generation from freshly prepared human leukocytes were also investigated. Compared to the filter sterilized PD-fluid, we found that heat treated PD-fluids significantly inhibited the growth of the two cell lines and impaired the stimulated release of TNF alpha and superoxide radicals. These results demonstrate that heat sterilization of PD-fluids produces substances that are cytotoxic regardless of the cell species, the cell type or the cell function tested.

In vitro toxicity of biomaterials determined with cell density, total protein, cell cycle distribution and adenine nucleotides.

Inhibition of cell growth is the most commonly used endpoint for in vitro toxicity of biomaterials. The use of several different endpoints might however generate more information concerning the nature of the toxicity. Thus, we examined the toxicity of two biomaterials, Polyvinylchloride (PVC) and Polyoximethene (POM), with different selected endpoints. The influence of cell growth on these endpoints was also investigated. Water extracts from the polymeric materials were tested on the continuous cell line L-929. Cell density, total protein, total protein per cell, fraction of cells in G0/G1- or S-phase, the concentration of ATP, ADP and AMP were used as endpoints. The PVC material did not significantly influence any of these endpoints until after 72 hours of exposure and the main part of the toxicity at 72 hours was related to higher proliferation rate in control cultures. After the cells had been incubated for 8 hour with POM the main toxic effect was on the energy parameters. In conclusion the PVC material was less toxic than the POM material. Our results also implies that the choice of endpoint will influence the evaluation of cytotoxicity.

Heat sterilization of fluids for peritoneal dialysis gives rise to aldehydes.

OBJECTIVE: To chemically identify and quantify glucose degradation products in heat sterilized fluids for peritoneal dialysis. DESIGN: Three different brands of commercial PD-fluids and one laboratory made fluid, sterilized either by heat or filtration, were investigated for the presence of aldehydes. MEASUREMENTS: Aldehydes were identified and quantified using high performance liquid chromatography and gas chromatography. RESULTS: The tested brands of heat sterilized PD-fluids were found to contain several different aldehydes while the sterile filtered PD-fluid contained none. The highest concentrations in commercial PD-fluids of these aldehydes were: acetaldehyde (420 microns), glyoxal (14 microns), methylglyoxal (12 microns) and formaldehyde (11 microns). Valeraldehyde was also identified but not quantified. The presence of 5-HMF (15 microns) and 2-furaldehyde (2 microns), which has been identified by others, was confirmed. CONCLUSIONS: The heat sterilization of commercial PD-fluids gives rise to several aldehydes which may contribute to adverse effects of PD-fluids on patients.

Toxicity of effluent peritoneal dialysis fluid.

Commercial heat-sterilized fluids for peritoneal dialysis (PD) are unphysiological due to low pH, high osmolality, and the presence of several toxic glucose degradation products, formed during heat sterilization. These properties are all believed to negatively affect host defense in patients. Both the low pH and the high osmolality are known to equilibrate after being introduced into the abdominal cavity of patients. Cytotoxicity due to glucose degradation products has, however, not yet been studied in this respect. Effluent peritoneal dialysates were collected from patients after dwell times of 0, 5, 15, 30, 120, and 240 minutes. Cytotoxicity, measured as the inhibition of cell growth of a cultured fibroblast cell line (L929), osmolality, and pH were determined. PD fluids, due to the presence of degradation products, were found to remain cytotoxic after a dwell time of more than 30 minutes. However, after 4 hours no cytotoxicity could be observed in the effluent fluids. Osmolality slowly decreased during the entire dialysis period, while pH rapidly increased and was close to neutral within 5 minutes following instillation. It is concluded that the presence of glucose degradation in PD fluids may be as important as low pH and high osmolality for clinical complications.

Toxicity of peritoneal dialysis fluids on cultured fibroblasts, L-929.

Peritoneal dialysis (PD) fluids are known to suppress the reactions of inflammatory cells in vitro. PD-fluids have also been shown to have cytotoxic influence on mesothelial cells. The combinations of these factors may have a detrimental effect on the peritoneum or may impair cellular defence against bacterial peritonitis. Some authors have discussed the relevance of heat sterilization to both so-called peritoneal side-effects and to chemical decomposition of fluids. Four commercial PD-fluids and one laboratory-made PD-fluid were tested for cytotoxicity on a cultured fibroblast cell line, L-929. Cytotoxicity was determined as an inhibition of cell growth by quantification of total protein. The laboratory-made PD-fluid was sterilized either by filtration or by filtration and heat. The commercial and the heat-sterilized laboratory made PD-fluids caused significant inhibition of cell growth (53 to 76%) in contrast to saline and the filter-sterilized laboratory-made PD-fluid. Since the pH values of all the testsolutions were neutral, low pH was not the cause of toxicity. Our results regarding the L-929 cells indicate that the cytotoxicity of PD-fluids is of a general nature. Furthermore, the results indicate that the heat sterilization process might be partially responsible for causing toxicity in PD-fluids.