|Publication||1Regional Blood Transfusion Center, National Blood Transfusion Service, Szeged, H-6701, P.O.Box 988, Hungary|
|Publication||2Department of Inorganic and Analytical Chemistry, University of Szeged, H- 6701, P.O.Box 440, Hungary|
|Publication||3GAMBRO Dialysis Center, Szeged, H-6725 Semmelweis u. 6, Hungary|
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Regeneration of Hemodialyzers with Ozone
The disinfection of hemodialyzer membranes with ozone was evaluated. Cellulosic membranes infected by Pseudomonas, Staphylococcus, Escherichia or Candida species could be sterilized very effectively by a 2-minute treatment with a 4 mg dm-3 ozone solution. Pyrogenic substances (endotoxins) can also be removed by ozone.
The effects of ozone on the permeability and mechanical properties of the membranes were assessed by determining the in vitro urea, creatinine and vitamin B12 clearances, the ultrafiltration coefficient and the pressure resistance. Treatment of the membrane with a 4 mg dm-3 ozone solution for 160 min caused no significant changes in these parameters.
These results indicate that ozone may be regarded as a possible disinfectant for hemodialyzer sterilization for reuse: it is a very effective germicide which does not lead to the formation of toxic disinfection by-products, and the functionality of the membrane remains intact during disinfection.
Dialyzer reuse is practiced in the treatment of end-stage renal disease in more than 75% of the dialysis units in the United States (1). Reuse practice is motivated primarily as an approach to reduce costs or to allow the use of more expensive hemodialyzers (2). It has been estimated that current annual savings from dialyzer reuse in the United States amount to more than $200 million (3). The performance of the dialyzer during reuse is affected by the deposition of blood elements inside the lumen of the blood compartment and onto the dialyzer membrane and by the interaction of the disinfectant and the membrane material. Accordingly, the functionality of the reused dialyzer depends on the nature of membrane material and on the germicide. The most frequently used disinfectants are sodium hypochlorite (bleach), what is used in conjunction with formaldehyde or glutaraldehyde, peroxyacetic acid in conjunction with hydrogen peroxide, and freshly heated citric acid (4). Concerns with dialyzer reuse include pyrogenic reactions, infections, toxicity from the disinfecting agent, and decreased
dialyzer performance for clearance and ultrafiltration (5). The chemical composition differs extremely widely with the type of dialyzer membranes, and their interactions with the blood elements and with reprocessing agents are likely to differ too. This may be the reason why the
b2-microglobulin clearance of reused membranes is decreased, remains unchanged or is increased, depending on the chemical composition of the disinfectant and on the structure of the membrane (6). The ultrafiltration coefficient of a polysulfone membrane increased by 41% when peroxyacetic acid was used, but did not vary with formaldehyde (7). The clearances of small and large solutes of reused high-flux dialyzers changed to different extents (8). It follows from the above facts that dialyzer reprocessing significantly impairs the dialysis delivery. Inasmuch as the patient survival in the chronic hemodialysis population is influenced by the clearances of small and middle-sized solute molecules, a precise knowledge of the membrane material and the reprocessing technique is important for the prescription of hemodialysis in centers practicing reuse. The ideal germicide for hemodialyzer reuse effectively sterilizes the membrane without changing its functionality. Since no such ideal disinfectant is known at present, there is an urgent need for the testing and introduction of new chemicals for dialyzer reuse. Ozone is is known to be a very effective disinfectant and antipyrogenic agent. It is used for the purification of dialysis water and its application for dialyzer disinfection has been patented (9, 10). However, few papers dealing with the use of ozone to disinfect hemodialyzers are to be found in the literature (11). The aim of the present work was to investigate the feasibility of the sterilization of hemodialyzer membranes with ozone, and to evaluate the effect of treatment on the functionality of the membrane.
Materials and Methods
Vitamin B12, creatinine hydrochloride and urea (SIGMA-ALDRICH Ltd., Hungary) were of analytical grade and were used without further purification. High-purity water was used for the preparation of the solutions. Ozone was generated from oxygen (99.95%, Linde) with a silent electric discharge in a home-made ozonizer. A Gambro Alwall GFE 12 high-flux cellulosic (cuprophanÒ) dialysis membrane was used for the experiments. The ozone concentration was determined via its absorbance at 260 nm. For calculation of the concentration, a molar absorptivity of e = 3000 M-1 cm-1 was used. The vitamin B12 concentration was determined via its absorbance at 362 nm (e = 26220 M-1 cm-1). The creatinine concentration was determined by the method of Jaffé (12) and the urea concentration by the method of Fawcett (13). The clearance was calculated according to the formula  (14):
Q (C – C )
C = Bi Bi Bo
D Bi C 
where CD is the clearance of the membrane (cm3 min-1), QBi is the flow rate (cm3 min-1) of the solution in the blood compartment, and CBi and CBo are the concentrations of the solute before and after the dialyzer, respectively.
The endotoxin concentration was determined by means of Chromogenic LAL assay (15). The microbiological measurements were performed by standard laboratory methods recommended by the WHO.
The experimental set-up for the ozonation of the membrane is outlined in Figure 1.
Figure 1. Experimental set-up for disinfection of hemodialyzer membrane with ozone (1 oxygen gas, 2 ozonizer, 3 ozone saturator, 4 water-phase UV ozone destructor, 5 catalytic gas- phase ozone destructor, 6 ozone detector, 7 peristaltic pump, 8 hemodialyzer membrane)
For investigation of the effect of ozone on the functionality of the membrane, the dialyzer was treated for a certain time with ozone-containing water, and the clearances of urea, creatinine and vitamin B12 were determined. The ultrafiltration coefficient was measured at transmembrane pressure of 27 kPa (200 mm Hg) (16).
Results and Discussion
An ideal chemical disinfectant for dialyzer reuse effectively sterilizes the membrane without changing the operating ability of hemodialyzer. When the sterilization affects the mechanical properties and functionability of the membrane, these changes have to be taken into consideration in the prescription of the hemodialysis treatment. Ozone, a very effective germicide with no toxic side-products, was earlier tested in vivo as a reagent for dialyzer reuse (11).
Before the disinfection experiments, the dialyzers were incubated with a mixed solution of Pseudomonas aeruginosa, Streptococcus aureus, Escherichia coli and Candida albicans (germ count >105 cm-3) for 12 h. The pretreated membranes were washed with ozone- containing deionized water and in parallel with ozone-free water (blank). After certain time intervals, samples were taken from the water and used for microbiological tests. Endotoxin concentrations in the samples were also determined. The washing time was 60 min. The membranes were next filled with the washing solution and stored at 10 oC for 24 h, after storage the sterility was tested.
Figure 2. Germ count of Pseudomonas aeruginosa as a function of the duration of washing with ozone solution ([O3] = 4 mg dm-3) and with ozone-free water (total flow rate 400 cm3 min-1 (250 cm3 min-1 in the blood compartment and 150 cm3 min-1 in the dialyzate compartment))
Figure 2 demonstrates that the washing of the membrane with ozone-free water was not effective in removing Pseudomonas aeruginosa, whereas treatment with a 4 mg dm-3 ozone solution caused perfect sterilization within 2 min. Analogous results were obtained with Escherichia coli and Staphylococcus aureus, but Candida albicans could be removed by 10- min washing with ozone-free water. These results can be explained by the fact that, in contrast with bacterial cells, Candida albicans does not attach firmly to the membrane. After storage for 24 h no microorganism could be detected in the water used to fill the ozone-treated membranes. Endotoxin concentrations were also measured in the solution used for the washing of the dialyzer membranes, as shown in Figure 3.
Figure 3. Endotoxin concentration as a function of the washing time. (The ozone concentration was 4 mg dm-3; the flow rate in the blood compartment was 250 cm3 min-1 and that in the dialyzate compartment was 150 cm3 min-1.)
The results revealed that ozone treatment is effective in the destruction of endotoxins too. The endotoxin level decreased to only about one-third of the starting concentration during ozone treatment for ~40 min, in spite of the fact that the sterilization was perfect after for ~2 min washing. As a reasonable explanation, it can be accepted that killed bacterial cells are adhere to the inner wall of the membrane, and endotoxins are continuously released from the cells during further ozone treatment.
The above results demonstrate that ozone is a very good antimicrobial agent, and an adequate antipyrogenic agent.
Investigation of operating ability of membrane
During disinfection, the operability of the dialyzer can be changed as an undesirable effect of the chemical interaction between the ozone and the membrane. The functionality of the dialyzers was characterized by measurement of the in vitro urea, creatinine and vitamin B12 clearances, the ultrafiltration coefficient and the pressure resistance. The results of the clearance test experiments are presented in Figures 4 and 5.
Figure 4. Vitamin B12 clearance as a function of the ozonation time ([O3] = 4 mg dm-3, flow rate in the blood compartment 250 cm3 min-1, and flow rate in the dialyzate compartment 150 cm3 min-1)
Figure 5. Urea and creatinine clearances as a function of the ozonation time ([O3]= 4 mgdm- 3, flow rate in the blood compartment 250 cm3 min-1, and flow rate in the dialyzate compartment 150 cm3 min-1
The clearances of urea, creatinine and vitamin B12 were not affected significantly by the ozone treatment of the membranes. It is important that the duration of this experiment (160 min) was much longer than the time necessary for disinfection (2 min; see above).
The ultrafiltration coefficients of the ozone-treated membranes were determined. There were no significant differences in comparison with the value given by the manufacturer (1200 cm3 h-1 at 200 mm Hg)
The mechanical stabilities of the dialyzers were tested by applying an intermembrane pressure of 101 kPa in the blood compartment (with closed dialyzate compartment). The dialyzate compartment was suddenly opened, and the magnitude of the transmembrane pressure change was observed. The membrane was considered to be damaged when a pressure decrease of >20 kPa could be observed. These results indicated that the ozone treatment did not impair the structure of the capillaries.
The results of these in vitro experiments demonstrate that ozone can be regarded as a good disinfectant in hemodialyzer reuse: it is a very effective disinfectant, and its chemical interaction with the membrane does not change the functionality of the dialyzer. It should be emphasized that these preliminary in vitro results must be supplemented by in vivo clinical investigations.
The applicability of ozone as a chemical agent for hemodialyzer reuse was investigated. It was found that ozone effectively and quickly sterilizes hemodialyzers and is able to remove pyrogenic endotoxins without influencing the mechanical properties and functionality of the membrane.
This work was supported by OMFB (Contract No. ALK- 00088/98) and by OTKA (Contract No. T029047). The financial support is highly appreciated.
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