Single circulation system

In this section a description is given of the single circulation system. This system was developed after to the previously described dual circulation sys-tem. The main difference of the single circulation system when compared to the dual circulation system was the absence of an enzyme circulation.

This was done to further decrease the risk of enzyme leakage. In the single circulation system (Figure 3-7, page 57) the glucose oxidase/catalase solu-tion was retained in an enzyme reactor. Isotonic saline was used to perfuse the microdialysis probe. A glucose eliminator was developed to prevent the accumulation of glucose in the perfusion system. An O₂-electrode was posi-tioned in line with the enzyme reactor to monitor the oxygen concentra-tion in the perfusion fluid. Before re-entering the microdialysis probe the saline was filtered by carbon filter to adsorb enzyme molecules that leaked out of the enzyme reactor. Placing a fluid equaliser after the pump reduced fluid pulsations caused by the piston pump. The pump, O₂-electrode, carbon filter and connective tubing used in this system and disinfection pro-cedures were the same as described in the section of the dual circulation sys-tem.

Microdialysis probe

The microdialysis probe used in this system was an enhanced version of the microdialysis probe described previously in the dual-circulation system sec-tion. The construction of the probe can also be subdivided in three stages and is outlined in figure 3-8, page 60.

Stage A:

Two dialysis tubes with a length of 20 mm were cut out a large bundle of dialysis tubes (Cellulose, Spectra/Por RC, i.d. 150 µm, o.d. 180 µm, 18,000 molecular weight cut-off) and glued (CA 1500) on respectively a 40 mm and 300 mm long silica tube (SGE Scientific Pty. Ltd., Sydney, Australia, i.d.

0.11 mm, o.d. 0.18 mm). Instead of the separate in- and outlet tubing used in the dual circulation probe, the in- and outlet of this probe were combined in one triple lumen tube (Polyethylene, Dural Plastics & Engineering,

Single circulation system

Auburn, Australia, i.d. 0.35mm, o.d. 1.0 mm, length 250 mm). The silica tube (300 mm), used as probe outlet, was threaded through one of the triple lumen (Figure 3-8A.1) leaving 30 mm of silica tubing free. Next the 40 mm silica tube, used as probe inlet, was positioned in one of the remaining two lumen (Figure 3-8A.2), leaving also 30 mm of silica tubing free. At both ends of silica the two dialysis tubes were glued (CA 1500). In the third lumen a tungsten wire (TW5-3, Clark Electromedical Instruments, UK, Ø 0.12 mm) was positioned to give the probe the required firmness as well as flexibility (Figure 3-8A.3). The two silica tubes and tungsten wire were sub-sequently fixed to the triple lumen tube with cyanoacrylate glue (CA 1500).

Stage B:

Once the glue had dried, a tube (Teflon, Zeus Industrial Products, i.d. 0.50 mm, o.d. 0.57 mm, length 30 mm) was slipped over both the dialysis tubes and the tungsten wire until it touched the triple lumen tube. Subsequently it was fixed with glue (CA 500). A second tube (Polyethylene, Rubber BV, i.d. 1.40 mm, o.d. 2.00 mm) was positioned over the interface of the triple lumen tube (Figure 3-8B.1) and the microdialysis probe and fixed with glue (CA 1500). At the tip of the microdialysis probe a turning point was placed (Figure 3-8B.2) as described previously in the dual-circulation system sec-tion, leaving in total 30 mm of dialysis tubing uncovered.

Stage C:

The final stage involved the fixation of a butterfly and tubing for the probe in- and outlet. For future fixation of the probe on the skin, a butterfly cut from a dwelling catheter (Venofix) was glued on the interface of the triple lumen tube and the microdialysis probe (Figure 3-8C.1). At the other end of the triple lumen tube connective tubing was fixed, serving as in- and outlet tubes. In one of the lumen, 20 mm of silica tubing was left uncovered and was used for the connection with the enzyme reactor (Figure 3-8C.2).

In the other lumen, used for the inlet of the microdialysis probe, a silica tube of 20 mm was fixed (CA 1500) and connected to a polyethylene tube (Rub-ber BV, i.d. 0.4 mm, o.d. 0.8 mm).

Single circulation system

The microdialysis probes were usually made in batches of five to ten and tested and stored as described previously.

Figure 3-8.Construction of the enhanced version of the microdialysis probe (stages A, B and C). For explanation of parts, see “Microdialysis probe” on page 58.

Enzyme reactor

An enzyme reactor was used for the enzymatic conversion of glucose recov-ered in the perfusion fluid (Figure 3-9A, page 62). For the exterior of the reactor, a 50 mm long stainless-steel tube (i.d. 0.5 mm, o.d. 0.70 mm) was used. Two dialysis tubes (Cellulose, Spectra/Por RC, i.d. 150 µm, o.d. 180 µm, 18,000 mwco) with a length of 60 mm were threaded trough this metal tube leaving 5mm of dialysis tube free on both sides. A turning point, made of a 5 mm long tube (Polyethylene, Rubber BV, i.d. 0.28 mm, o.d.

0.61 mm) with one end previous sealed (CA1500), was fixed on one side of the two dialysis tubes with glue (CA 1500) leaving a minimum of death vol-ume. Two tubes (Silica, SGE Scientific Pty. Ltd., i.d. 0.11 mm, o.d. 0.18 mm, length 20 mm) necessary for filling of the reactor with enzyme

solu-(C)

Single circulation system

tion, were pushed in for 10 mm on both sides of the metal tube. Next the dialysis- and silica tubes were carefully glued to the metal tube which had both ends made rough for better glue fixation. Both open ends of the dial-ysis tubes were glued to connective tubing (Polyethylene, Rubber BV, i.d.

0.28 mm, o.d. 0.61 mm) for placement in line with the microdialysis probe and the oxygen electrode. After drying of the glue the reactor was tested by flushing with air and water. Before filling with enzyme solution, the reactor was rinsed with ethanol (70%) and sterile water.

Glucose eliminator

Not all the recovered glucose is converted in the enzyme reactor. Therefore a glucose eliminator was developed to prevent accumulation of glucose within in the perfusion system. Elimination of glucose from the perfusion fluid by the eliminator is based on the enzymatic conversion of glucose by god. Both its functioning and design are based on the glucose reactor. The eliminator was made of two dialysis tubes (Cellulose, Spectra/Por RC, i.d.

150 µm, o.d. 180 µm, 18,000 mwco) with a length of 160 mm lying side by side in a Teflon tube (Zeus, i.d. 0.70 mm, o.d. 1.50 mm, length 150 mm) with one end of the dialysis tube fixed in a turning point (Polyethylene, Rubber BV, i.d. 0.28 mm, o.d. 0.61 mm, length 5 mm, one side sealed).

Two tubes (Polyethylene, Rubber BV, i.d. 0.28 mm, o.d. 0.61 mm, length 30 mm) necessary for filling the eliminator with enzyme solution, where each positioned on both sides of the Teflon tube. The dialysis tubes and enzyme filling tubes were fixed in the Teflon tube with glue (CA 1500).

After drying each of the dialysis tubes open ends were glued (CA 1500) to connective tubing (Polyethylene, Rubber BV, i.d. 0.40 mm, o.d. 0.80 mm).

Upon use, the glucose eliminator was rinsed with an ethanol solution (70%) and sterile water after which the eliminator was filled with enzyme solution.

Equaliser

The dialysis flow should be regular because it is an important parameter in the recovery of substances. Changing flows will result in variable recoveries.

Protracted fluid pulsation may deform the ultra-filtration membranes used

Single circulation system

in the carbon filter, resulting in an increase in flow and thus in a change in recovery. To reduce the effect of fluid pulsation an equaliser was constructed and inserted in the perfusion system after the piston pump (Figure 3-9B). It was made of a glass tube (i.d. 3.5 mm, o.d. 4.0 mm, length 75.0 mm) where one end was sealed. Two connective tubes (Polyethylene, Rubber BV, i.d.

0.40 mm, o.d. 0.80 mm) were glued (CA 1500) into the glass tube opening.

When the equaliser was inserted in the perfusion system an air bubble was trapped in the dead end of the glass tube and consequently absorbed the fluid pulse caused by the piston pump. Before use the equaliser was rinsed with an ethanol solution (70%) and sterile water.

Figure 3-9.Construction of the enzyme reactor (A) and equaliser (B).

Enzyme solution

The preparation of the enzyme solution used in this system was the same as described in the dual-circulation system section except for the amount of glucose oxidase used. Instead of the 1,000 units god/ml present in the enzyme solution of the dual circulation system, 10,000 units god/ml were used to fill the enzyme reactor and glucose eliminator.

Stainless-steel tube

Enzyme filling point Enzyme filling

point

Dialysis tube Enzyme solution Inlet

Outlet

Air bubble Glass tube

Inlet Outlet

(A)

(B)

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