Abstract
Carbohydrates are not stable when exposed to energy; they degrade into new molecules. In peritoneal dialysis (PD) fluids, degradation of glucose occurs during the heat sterilization procedure. The biological consequences of this degradation are side effects such as impaired proliferation and impaired host defense mechanisms, demonstrated in vitro for a great variety of cells.
Several highly toxic compounds—such as formaldehyde and 3-deoxyglucosone—have been identified in PD fluids. Carbonyl compounds, apart from being cytotoxic, are also well-known promoters of irreversible advanced glycation end-products (AGEs), which might participate in the long-term remodeling of the peritoneal membrane.
Various approaches can be used to reduce the formation of glucose degradation products (GDPs) during heat sterilization. Some examples are shortening the sterilization time, lowering the pH, removing catalyzing substances, and increasing glucose concentration. The latter three factors are employed in the multi-compartment bag with a separate chamber containing pure glucose at high concentration and low pH.
Gambrosol trio, a PD fluid produced in this way, shows reduced cytotoxicity, normalized host defense reactions, less AGE formation, and reduced concentrations of formaldehyde and 3-deoxyglucosone. Moreover, in the clinical situation, the fluid turns out to be more biocompatible for the patient, causing less mesothelial cell damage, which in the long term could lead to a more intact peritoneal membrane.
Conclusion
Glucose degradation products in heat-sterilized fluids for peritoneal dialysis are cytotoxic, promote AGE formation, and cause negative side effects for the patient. Using improved and well-controlled manufacturing processes, it is possible to produce sterile PD fluids with glucose as the osmotic agent but without the negative side effects related to GDPs.
One of the best known glucose degradation products (GDPs) from the field of parenteral solutions is 5-HMF (5-hydroxymethylfuraldehyde). The limit set in various pharmacopeias for the presence of 5-HMF is much higher than the concentrations typically found in PD fluids. From a clinical point of view, 5-HMF in parenteral solutions is not considered a significant toxicological problem (1). However, other and more toxic degradation products—such as a wide range of acids and carbonyl compounds—have also been reported in parenteral solutions (2-4).
In peritoneal dialysis, an increasing number of reports are quantifying glucose degradation products. These products are coming under investigation because the PD patient is continuously exposed to heat-sterilized glucose-containing solutions. The glucose degradation products currently identified and quantified in PD fluids are fructose, formaldehyde, acetaldehyde, methylglyoxal, glyoxal, 5-HMF, 2-furaldehyde, and 3-deoxyglucosone (5,6). These degradation products may, however, represent only a small fraction of a likely larger number of degradation products that could be generated from glucose. It is important to note that the most reactive and toxic substances present in PD fluids have not yet been identified (7).
Biological Activity of GDPS in the Acute or Short-Term PD Perspective: In Vitro and in Vivo Evidence
The biological reactivity of glucose-derived degradation products involves cytotoxicity, modulation of cellular signal pathways, and protein modification by cross-linking. The biological reaction pattern of GDPs in PD fluids, as assessed in vitro, was first demonstrated as strongly reduced proliferation of a cultured mouse fibroblast cell line, L-929 (8,9). This cell line represents an internationally well-proven, well-characterized, and reproducible in vitro system, often used as first-line screening for basal cytotoxicity properties.
Recently, the use of primary cultures of human peritoneal mesothelial cells has revealed an almost identical level of cytotoxicity as compared with the fibroblast cell line (10). However, the use of mesothelial cells under certain experimental conditions represents a more sensitive system than the use of immortalized tumor cell lines. Thus, the cytotoxic activity of GDPs detected in human mesothelial cells emphasizes biological relevance in peritoneal dialysis. The in vitro findings for mesothelial cells have been confirmed in recent clinical studies, which have shown a more intact and obviously regenerable mesothelial cell layer on the peritoneal membrane when a new PD fluid with less GDPs is used. After just 1 month of treatment, a more than 2-fold increase in cancer antigen 125 (CA125) concentration was found in the effluent dialysate, which undoubtedly indicates a significantly increased mass of mesothelial cells (11,12).
Not only have acute cytotoxic effects been ascribed to GDPs, but also an influence on various cellular host defense reactions: that is, impaired cytokine and oxygen radical release in vitro (10,13-16), and modulated interaction between leukocytes and endothelial cells in vivo (17). Furthermore, clinical evidence clearly links GDPs with the occurrence of 21 cases of chemical peritonitis (18). According to the report, the problem was caused by a batch of PD fluid that had been produced at an inappropriate pH; the resulting fluid contained notably high concentrations of GDPs.
When it comes to acute effects of GDPs on transport characteristics, the combination of low pH and GDPs has been shown, in a rat model of PD, to cause a slight increase in small-solute transport, indicating that an increased surface area is initially available for solute exchange (19). A short-term clinical study (20) has also demonstrated a marked loss of ultrafiltration capacity with older PD fluids (higher concentrations of GDPs) compared to freshly produced PD fluids (lower concentration of GDPs).
Several direct and indirect evidences of acute cell interaction or activation potential of GDPs are still to be found. The exact mechanisms at the molecular level are unknown, but reactive carbonyl groups are known to modulate many biological signal pathways.
Biological Side Effects of GDPS in Long-Term PD
Local Effects in Peritoneal Tissue
Ultrafiltration failure, owing to increased permeability of glucose, is a prominent cause of withdrawal from PD. The underlying pathophysiological mechanism has so far not been explored in detail. Advanced glycation end-products (AGEs) are known to accumulate in the vascular walls and in other tissues. They are suggested to play a significant role in the long-term increase in peritoneal membrane permeability (21,22).
Advanced glycation end-products are a heterogeneous group of molecules with a variety of biological activity; they are formed when a carbonyl group of a reducing sugar condenses with a reactive amino group of a protein. Until now, AGE formation in PD has been linked mainly to the use of glucose as an osmotic agent. However, GDPs are now recognized as much stronger promoters of AGEs than glucose per se (23,24). The presence of various reactive carbonyl species, as in PD fluids, can modulate the reaction pathway in the cascade from Schiff base and Amadori product to irreversible AGE products.
Growth factors such as vascular endothelial growth factor (VEGF), a powerful angiogenic factor, could also be a link to the long-term permeability changes observed in PD patients. Glucose degradation products are known to induce VEGF through cross-linking of endothelial and mesothelial cell surface proteins (25). Enhanced production of VEGF by peritoneal cells might stimulate angiogenesis and increase the available vascular surface area, resulting in an increased small-solute permeability.
Peritoneal dialysis per se creates a local intraperitoneal situation of diabetes, which is dramatically enhanced by the presence of carbonyl stress compounds in the PD fluid. It should be emphasized that the molecular events behind dialysis-induced functional and morphological alterations of the peritoneal membrane remain unknown. However, everything points to GDPs as the most important of the PD fluid components involved in the process of peritoneal membrane deterioration.
Systemic Effects
Advanced glycation end-products are suggested to be potential uremic toxins; they are known to accumulate over time as a result of reduced excretion and increased generation owing to permanent carbonyl and oxidative stress (26). The most well-known promoters of AGE formation are various dicarbonyl compounds such as 3-deoxyglucosone (3-DG), methylglyoxal, and glyoxal, all of which are present as GDPs in PD fluid (5,6,27). Of these, 3-DG is the main dicarbonyl, being present in concentrations between 100 – 200 μmol/L. For standard PD treatment, this concentration corresponds to giving a patient more than 100 g of 3-DG annually.
What happens with the infused 3-DG?
Earlier studies have shown that 3-DG disappears in a time-dependent manner from the effluent peritoneal dialysate (27,28). The reason could be that the infused 3-DG immediately reacts with and cross-links proteins of the peritoneal membrane and can, therefore, no longer be found in the dialysate. Alternatively, 3-DG might be transported from the peritoneal cavity across the capillaries to the systemic circulation. This later explanation is supported by the fact that 3-DG disappears from the dialysate at approximately the same rate as glucose. The molecular sizes of 3-DG and glucose are very close; 3-DG would theoretically have a slightly higher clearance out of the peritoneal cavity if no reaction on the peritoneal membrane occurs. The permeability–surface area product [PS, mass transfer area coefficient (MTAC)] for glucose transported from the peritoneal cavity to plasma is usually set at 15.4 mL/min during three-pore simulations (29), while the PS for 3-DG is 16 mL/min. This PS value corresponds well with measured values, as shown in Figure 1. Transport of 3-DG from the PD fluid across the membrane to the blood side is further supported by the observation of a sharp increase in plasma 3-DG after 30 minutes of acute PD in rats (Figure 2).
Calculated intraperitoneal concentrations of 3-deoxyglucosone [3-DG (solid line)] compared to measured values (closed circles), mean ± standard error of mean. The first point was measured in the fresh dialysis fluid before instillation into the peritoneal cavity; it was corrected for a residual volume of 300 mL. The mass transfer area coefficient [(MTAC), permeability–surface area product (PS)] for 3-DG (16.0 mL/min) in this example was slightly higher than that for glucose (15.4 mL/min) to compensate for the smaller molecular radius of 3-DG.
A rat model of acute peritoneal dialysis was used to study 3-deoxyglucosone (3-DG) in plasma during a 4-hour dwell. Anesthetized rats were infused intraperitoneally with 20 mL conventional peritoneal dialysis fluid containing 2.5% glucose, and blood samples were collected for analysis of 3-DG. Results are shown as mean ± standard error of mean (n = 4).
We speculate that, for patients treated with peritoneal dialysis, the continuous load of 3-DG from the dialysis fluid might be another component contributing to enhanced oxidative and carbonyl stress and consequently to local and systemic accumulation of AGEs.
Can the 3-DG Load for PD Patients be Decreased?
Gambrosol trio, a new PD fluid optimized to reduce GDPs, is produced in a three-compartment bag. Two separate chambers contain pure concentrated glucose at very low pH. Thus, the heavy degradation of glucose during heat sterilization is avoided, and the glucose is protected from further degradation during storage. Apart from causing very low AGE formation in vitro, Gambrosol trio contains extremely low levels of 3-DG. As seen in Table 1, the 3-DG content is lowered from 97 – 170 μmol/L (in conventional PD fluids) and 34 – 70 μmol/L (in two-compartment bags) (30,31) to just 18 μmol/L in the three-compartment bag (6). This very low concentration in Gambrosol trio is known to remain stable during more than 18 months of storage. Thus, exposure to 3-DG from peritoneal dialysis could be reduced by approximately 90% by using a well controlled PD-fluid manufacturing process, as in the case of Gambrosol trio.
Reported Concentrations a of 3-Deoxyglucosone (3-DG) in Various Peritoneal Dialysis Fluids
Concentrations of 3-DG were measured for mixed, ready-to-use solutions with a final glucose concentration of 1.5%. The concentration of 3-DG in the three-compartment bag was analyzed using reverse-phase high-performance liquid chromatography with diaminonaphthalene as derivatization agent (6).
Glucose degradation products are highly reactive substances. Because there is absolutely no therapeutic benefit to the presence of these compounds in PD fluids, they should be removed. The conclusion seems obvious: the time has come to prevent infusion of GDPs into the peritoneal cavity, especially when affordable technologies or technical solutions are available.
Conclusions
We conclude that glucose degradation products in heat-sterilized fluids for peritoneal dialysis are cytotoxic in the short term. Over the long term, GDPs— in particular, 3-deoxyglucosone—increase carbonyl stress locally and systemically. Using improved manufacturing processes, it is possible to produce sterile PD fluids with glucose as osmotic agent but without the negative side-effects related to GDPs.
Footnotes
Acknowledgment
Special thanks to Randi Ipsen for valuable comments on the manuscript.