**1. Introduction**

196 Progress in Hemodialysis – From Emergent Biotechnology to Clinical Practice

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704.

In the dialysis clinic, water is an essential vehicle to deliver life-saving treatment to patients suffering from varying degrees of kidney failure, both acute and chronic. Clean water is vital, as the key ingredient used to prepare hemodialysis fluid (dialysate solution), and online generation of substitution fluid for hemodiafiltration. Generally all fluids used to treat patients suffering from kidney failure may come into contact with the blood of the patient, whether directly or indirectly (across a membrane), and theoretically could transport contaminants resulting in a negative impact on patient health. Of the microbiological contaminants found in water, endotoxin is given considerable attention, given its difficulty for removal and inactivation from water and water distribution systems (Smeets et al., 2003; Perez-Garcia & Rodriguez-Benitez, 2000) and its inherent pyrogenicity (G. Lonnemann, 2000).

Endotoxins are found in all gram-negative bacteria, although slight differences in chemical structure are found between varying bacterial strains. The term endotoxin is typically used to describe a complex of protein and lipopolysaccharide (LPS) molecules found in the outer cell wall of gram-negative bacteria, that either slough off during growth, or are released upon cell lysis. Endotoxin and lipopolysaccharide are typically used interchangeably in literature, although in clinical discussion the term endotoxin is most often used, as it is the metric used to monitor water and dialysis fluid quality. Lipopolysaccharide is a vital component of the outer membrane of gram-negative bacteria, providing numerous physiological functions and comprising nearly 75% of the bacterium outer surface area (Raetz, 1991). Lipopolysaccharides consist of three components: a long heteropolysaccharide chain (O-specific chain) which represents a surface antigen; a core oligosaccharide; and a lipid component termed lipid A used as an anchor in the outer cell membrane (Rietschel et al., 1994; Gorbet & Sefton, 2005). Molecular weights of most lipopolysaccharides are 10 – 20 kDa; however, due to their amphiphilic nature, LPS molecules can form aggregates (100 – 1000 kDa) which are too large to pass through dialysis membranes. It has been shown that components of lipopolysaccharide (lipid A) are able to pass through dialysis membranes, can elicit a pyrogenic response (Naveh-Many et al., 1999), and contribute to long-term morbidity and inflammation (H. Schiffl, 2000; Raj et al., 2009).

Lipid A is the most conserved component of lipopolysaccharide throughout all gramnegative bacteria, and as such is responsible for the majority of the pyrogenic activity. Lipid A consists of a phosphorylated N-acetylglucosamine (NAG) dimmer connected to saturated

Dialysis Membrane Manipulation for Endotoxin Removal 199

polymer modifications, as well as chemical changes to the membrane composition, have been investigated to ascertain their influence on preventing or minimizing endotoxin and bacterial fragment flux across the membrane. Some studies have shown that even the choice of sterilization modality may have an impact on the membrane, and affect the ability to retain endotoxin (Gomila et al, 2006; Krieter et al., 2008). It is necessary to understand how a particular dialysis membrane interacts with endotoxin and other dialysis fluid contaminants, as the membrane is the last barrier to the patients' blood. Endotoxins (and other types of microbial contamination) are removed from dialysis fluid mainly by one of two methods: filtration and adsorption. Studies have shown that both methods of endotoxin removal occur during dialysis treatment (Osumi et al., 1995). Understanding endotoxin interactions with various membrane surfaces is imperative in order to orchestrate changes that will have a positive impact on endotoxin removal. The end goal of all endotoxin research is to limit patient exposure, in hopes of reducing the chance for pyrogenic

Prior to synthetic membranes occupying the majority of the dialysis filter market, cellulosic membranes were the predominant choice for manufacturers. Cellulose was a material that could be modified to improve its biocompatibility; however its geometrical manipulation was limited due to the production process. As membrane materials progressed from cellulose-based to synthetic, numerous adjustments could be made to the physical

One of the most direct methods to inhibit endotoxin is to change the material structure of the membrane itself, as the physical attributes of the membrane will perhaps have the greatest effect on endotoxin removal. Typically a thin-wall membrane is not as robust as a thick-wall membrane, in terms of endotoxin adsorption, since a thicker membrane can offer more surface area for the endotoxin to come into contact with. A thicker membrane provides a longer path for the endotoxin to maneuver through, before it reaches the blood circuit. An important characteristic of this path from outer membrane surface to inner membrane lumen is tortuosity, the curving path that the endotoxin must follow in order to reach the blood compartment of the fiber membrane (Osumi et al., 1995). As tortuosity is increased, the greater the chance the membrane has at prohibiting the passage of endotoxin. Membrane geometry changes can lead to differences in the adsorptive capacity for endotoxin (Vanholder & Pedrini, 2008; Vaslaki et al., 2000) and for bacteria (Waterhouse &

Changes in membrane permeability are controlled to enhance convective transfer, which targets middle molecular removal of species such as β2Microglobulin and vitamin B12. As dialysis membranes are pushed for more convective removal ability, the opportunity for endotoxin trans-membrane flux increases due to the higher chance of back-filtration. Future membranes designed to address middle molecule removal by increasing internal filtration (Mineshima et al., 2009) will not improve on patient inflammation (Kerr et al., 2007) unless

The effect of membrane thickness and permeability on endotoxin removal was studied by testing various membrane configurations, and by observing their ability to restrict contaminant flux. Synthetic membranes for testing were manufactured with specific geometries to observe their performance relative to a control. Fiber geometries tested included low flux, high flux, thin wall, thick wall, macrovoid, and a control. Characteristics

membrane geometry is modified to improve endotoxin removal.

characteristics of the membrane by relatively simple manufacturing process changes.

reactions, inflammation, and shock.

**2.1 Membrane geometry** 

Hall, 1995).

fatty acid chains; variability within the composition of the fatty acids will determine the toxic property of lipid A, as well as play a role in resistance to host antimicrobial factors and avoiding recognition from specific components of the host immune system (Bland et al., 1994; Gunn, 2001; Qureshi et al., 1999). The O-specific side chain component of LPS is responsible for complement activation and contributes to fever and hypotension, as well as binding to endotoxin recognition molecules within the body (Valvano, 1992; Bailat et al., 1997). Once within the body, LPS tend to be found at higher concentrations within the spleen and liver (uptake by phagocytosis) where they are cleared from the body (Haeffner-Cavaillon et al., 1998).

Dialysis patients are typically exposed to 90 – 120 liters of dialysis fluid per treatment, which equates to an annual exposure of 20 – 30,000 liters (Weber et al., 2004; I. Ledebo, 2002). With constant exposure to large amounts of fluid, the opportunity for a dialysis patient to experience an inflammatory or pyrogenic reaction due to contamination within the dialysis fluid is increased. For hemodialysis, fluids that are used for treatments do not have to be sterile; however, the lower the microbial concentration, the lower the risk of patient reaction. Because of this risk, regional regulatory boards have implemented limits to the total microbial count that can be present in fluids that are to be used in dialysis treatments. However, even if water treatment systems are in place, contamination is still a possibility and a risk. Dialysis fluid used for clinical treatments may become contaminated from either the source water, the dialysate concentrate, or from the water distribution system. Due to the ubiquitous nature of biofilm, and its propensity to generate endotoxin, this problem affects not only hemodialysis, but all extracorporeal therapies (Kanagasundaram et al., 2009).

Regardless of the treatment processes used to create water for dialysis fluid, the final opportunity to remove microbial contaminants from the fluid path prior to patient exposure is the dialysis membrane contained within the dialyzer. Dialysis fluid comes into direct contact with this membrane, and due to transmembrane pressure differences and the permeability of the filter, especially for high-flux dialyzers, the potential for dialysis fluid to enter the blood compartment and return to the patient is significant (N. Hoenich, 2007). It is this final barrier, the dialyzer membrane, where the last opportunity resides to remove endotoxins from solution (Weber et al., 2009), by means of membrane manipulation. The aim of the following research is to achieve a more thorough understanding of how endotoxin interacts with various physical characteristics of the dialysis membrane, and how to exploit these interactions to increase endotoxin removal from dialysis fluid.
