Degradation in peritoneal dialysis fluids may be avoided by using low pH and high glucose concentration.

OBJECTIVE: When glucose is present in a medical fluid, the heat applied during sterilization leads to degradation. The glucose degradation products (GDPs) give rise to bioincompatible reactions in peritoneal dialysis patients. The extent of the degradation depends on a number of factors, such as heating time, temperature, pH, glucose concentration, and catalyzing substances. In the present work, we investigated the influence of pH and concentration in order to determine how to decrease the amounts of GDPs produced. DESIGN: Glucose solutions (1%-60% glucose; pH 1-8) were heat sterilized at 121 degrees C. Ultraviolet (UV) absorption, aldehydes, pH, and inhibition of cell growth (ICG) were used as measures of degradation. RESULTS: Glucose degradation was minimum at an initial pH (prior to sterilization) of around 3.5 and at a high concentration of glucose. There was considerable development of acid degradation products during the sterilization process when the initial pH was high. Two different patterns of development of UV-absorbing degradation products were seen: one below pH 3.5, dominated by the formation of 5-hydroxy-methyl-2-furaldehyde (5-HMF); and one above, dominated by degradation products absorbing at 228 nm. 3-Deoxyglucosone (3-DG) concentration and the portion of 228 nm UV absorbance not caused by 5-HMF were found to relate to the in vitro bioincompatibility measured as ICG; there was no relation between 5-HMF or absorbance at 284 nm and bioincompatibility. CONCLUSION: In order to minimize the development of bioincompatible GDPs in peritoneal dialysis fluids during heat sterilization, pH should be kept around 3.2 and the concentration of glucose should be high. 5-HMF and 284 nm UV absorbance are not reliable as quality measures. 3-DG and the portion of UV absorbance at 228 nm caused by degradation products other than 5-HMF seem to be reliable indicators of bioincompatibility.

Glucose degradation products in peritoneal dialysis fluids may have both local and systemic effects: a study of residual fluid and mesothelial cells.

OBJECTIVE: When peritoneal dialysis (PD) fluids are heat sterilized, glucose is degraded to carbonyl compounds. These compounds are known to interfere with many cellular functions and to promote the formation of advanced glycation end-products. However, little is known about what actually happens with glucose degradation products (GDPs) after infusion into the peritoneal cavity. The aim of the present study was to investigate possible targets for GDPs in the peritoneal cavity. DESIGN: In vitro reactions between residual fluid and GDPs were studied by incubating unused PD fluid with overnight dialysate. Confluent monolayer cultures of human mesothelial cells were used as a model to study the reactions of GDPs with the cells lining the peritoneal cavity. METHODS: Samples were analyzed, using high pressure liquid chromatography, for the presence of formaldehyde, acetaldehyde, 5-hydroxymethyl-2-furaldehyde (5-HMF), methylglyoxal, and 3-deoxyglucosone (3-DG). Cytotoxicity was determined as inhibition of proliferation of cultured fibroblasts. RESULTS: None of the analyzed GDPs reacted with overnight dialysate. Formaldehyde and methylglyoxal, in contrast to 3-DG and 5-HMF, reacted with the cultured mesothelial cells. CONCLUSIONS: Low molecular weight carbonyls such as formaldehyde and methylglyoxal most probably react with the mesothelial cells lining the peritoneal cavity, and could be responsible for the disappearance of these cells during long-term treatment. 3-Deoxyglucosone showed remarkably low reactivity and was most probably transported within the patient.

In vitro cytotoxicity of four different buffers for use in peritoneal dialysis.

Various buffers can be used in fluids for peritoneal dialysis (PD). Lactate is the most frequently used buffer, but bicarbonate and pyruvate have been suggested as more biocompatible alternatives. In the past, acetate was used as a buffer in PD fluids, but was abandoned after being linked with sclerosing peritonitis and loss of ultrafiltration. When a new buffer for PD fluids is introduced, one of its most important characteristics is that it must be non-toxic, i.e. that it does not influence fundamental cellular functions. The aim of this study was to investigate the basal cytotoxicity of bicarbonate, acetate, lactate and pyruvate at neutral pH. As target cells, we used cultured mouse fibroblast-like L-929 cells, a well-known cell line approved by the authorities for regulatory use, and primary human mesothelial cells, which are the cells that line the peritoneal cavity and are exposed to the PD fluid in vivo. Pyruvate was more cytotoxic than lactate and bicarbonate, and no significant difference in cytotoxicity was found between lactate and bicarbonate. The human mesothelial cells were more sensitive to exposure to pyruvate than the L-929 fibroblast-like cells, but less sensitive to exposure to pure PD fluids. Thus, we recommend that both types of cell are used for the evaluation of the biocompatibility of PD fluids.

Pharmacologic importance of the reversible fatty acid conjugation of budesonide studied in a rat cell line In vitro.

Functional implications of the recently described fatty acid conjugation of budesonide (BUD) (Tunek, A., K. Sjödin, and G. Hallström, Drug Metabol. Dispos. 1997;25:1311-1317; Miller-Larson, A., E. Hjertberg, H. Mattsson, M. Dahlbäck, A. Tunek, and R. Brattsand, Am. J. Respir. Crit. Care Med. 1997;155:A353 [Abstr.]) were studied in a rat cell line, Rat1, transfected with the activation protein-1 (AP-1)-controlled regulatory element (TRE) driving the reporter gene beta-galactosidase. TRE is downregulated by glucocorticosteroids (GCS) through interaction with the AP-1 complex. BUD was compared to fluticasone propionate (FP), a potent glucocorticosteroid that does not form fatty acid conjugates. The kinetics and metabolism of the GCS were studied after incubation of either 3H-BUD or 3H-FP with transfected Rat1 cells. Up to 20% of added BUD was taken up into the cells over 24 h. The great majority of the intracellular radioactivity (80-90%) consisted of lipophilic BUD conjugates. After removing extracellular 3H-GCS, the outflow of radioactivity was studied. Only free BUD and not fatty acid conjugates was detected extracellularly, suggesting that hydrolysis of the conjugates was required to release BUD from the cell. During 165 min, less BUD (about 65% of totally incorporated) was released than FP (more than 90%). In the functional studies, FP was about six times more potent than BUD in downregulating TRE after 24 h continuous exposure. However, after a 6-h pulse of GCS, the effect of BUD persisted unchanged 18 h later, whereas FP had almost lost its efficacy (P < 0.05 between the drugs). In addition, the reversible conjugation process of BUD resulted in transferable GCS effects. Medium containing released BUD from previously loaded cells mediated nearly the same downregulatory effect after addition to naive cells as did continuous treatment. No such transferable effect was seen for FP. In conclusion, the reversible fatty acid conjugation of BUD resulted in prolonged cellular retention and anti-inflammatory activity after pulse exposure in this in vitro system. This fatty acid conjugation mechanism appears to add to the beneficial pharmacologic profile of BUD.

Toxicity of 20 Chemicals from the MEIC Programme Determined by Growth Inhibition of L-929 Fibroblast-like Cells.

The Multicentre Evaluation of In vitro Cytotoxicity (MEIC) programme is an international project aimed at evaluating the relevance of in vitro tests in predicting human toxicity. We have screened 20 chemicals (MEIC codes 31-50) from the programme, by using a cytotoxicity test based on growth inhibition of the mouse fibroblast-like L-929 cell line. Inhibition of cell growth was determined by the neutral red uptake method, which is well established and is used for screening the cytotoxicity of chemicals and plastics for pharmaceuticals and medical devices. The concentrations causing 50% inhibition of cell growth after a 72-hour exposure period varied from 3.1µM for hexachlorophene, to 1.4mM for caffeine. This is within the same range as results recently obtained with five other cell models. However, with some chemicals (chloroform, carbon tetrachloride and dichloromethane), no reliable results were obtained. These substances could not be dissolved in a reproducible way in any of the solvents used and, furthermore, they were highly volatile, which led to difficulties in maintaining the concentrations.