Urea/f-BSA/cytochrome c concentrations in retentate after dialysis: 12 hours

Continuing on the path of hemodialysis, we are increasing the complexity of the fluid being dialyzed. In this long-term experiment I will pump 100% serum with added urea (1mM) cytochrome c (1 mg/mL) and fluorescent BSA (1 mg/mL). So I need to detect urea, BSA, and cytochrome c in serum. Cytochrome c absorbs at 405 nm. The oxired in the urea assay kit absorbs at 570 nm, so I’m using FITC tagged BSA, (exitation 492 nm, emision 520 nm). TRITC emits at 570 nm so would interfere with urea detection.

In 5 mL of 100% serum I mixed…
0.3 g urea (1 mM concentration)
0.005 g cytochrome c (1 mg/mL)
0.005 g f-BSA (1 mg/mL)

The setup was the same as <this  post> with isopropanol followed by PBS pumped for 12 hours before flowing the Serum at 5.6 µL/min for > 12 hours with collections taken at half hour intervals. The chips are untreated after the post anneal in the RTP.

I used the Tecan to measure the serum retentate. The expectation is the the albumin will maintain its initial concentration (or increase in concentration due to ultrafiltration). The cytochrome c and urea are expected to decrease in concentration due to dialysis.

I was not able to detect the urea in the presence of the other analytes so have plotted the urea concentration from previous experiments. The assay kit claims to be able to detect urea in blood so I don’t suspect the cytochrome c or the BSA by itself but the f-BSA may be blocking detection of the urea. The following plot shows the initial concentrations for urea, cytocrhome c and f-BSA as 1 mM, 1 mg/mL , and 1 mg/mL respectively. The f-BSA remains at its initial concentration through the 12 hour test period. The cytochrome c shows a reduction, but not as much as the urea (from a previous experiment).

The collection tubes were weighed before and after collection. From this and the density of serum (1.025 g/mL) I calculated the volume collected for each half hour. The average sample taken was 132.88 µL, this is a rate of 4.43 µL/min. The syringe pump was set to pump 5.6 µL/min. The pump rate was not measured but taken as set. This difference leave us with a 20% Ultrafiltration rate. This assumes fluid is not lost elsewhere (e.g. leaks in the device or test set-up). The problem with this is that if water volume is being lost due to ultrafiltration, the concentration of albumin should go up. I removed the device from the beaker after the test and pulled alcohol through the device without any leaking. perhaps the 3-way valve is leaking.

I would like to run tagged BSA and cytochrome c with out the urea. Then I can use the TRITC tagged albumin that Tucker has been making since I wouldn’t have to worry about the interference from the oxired probe. I would also like to begin using a counter flow device making it possible to detect analytes in the filtrate as well as the retentate.