ANALYTICAL
BIOCHEMISTRY
94,109- 111 (1979)
Improved
Microdialysis
Technique
PAUL H. BRANDANDRACHELSTANSBURY Department of Physiology, Medical College of Ohio, CS #10008~ Toledo, Ohio, 43699 Received September 13, 1978 A simple, inexpensive method is described for dialysis of microliter amounts of aqueous samples against large volumes of solution with complete recovery of the fluid dialyzed. An example is given of application of the method to separation of [“Hlinulin from a monosaccharide.
pressure. To use, the sample is added, the upper surface covered with a greased coverslip, and, to commence dialysis, the chamber is placed on the dialysis wick [equivalent to the glass-wool pad, Ref. (1, Fig. Id)]. Other differences from Awdeh’s method are that in order to retain inulin (M, - 5500) we selected a dialysis membrane rated to retain substances of molecular weight ~3500 (A. H. Thomas); for convenience, a soft filter-paper strip (cut from a thin-layer chromatographic saturation pad, A. H. Thomas) was substituted for the glass-wool pad. To test the efficacy of the microchamber in separating [3H]inulin from small molecular weight compounds, we prepared a solution of 42 dpm nl-’ [3H]methoxyinulin (speMETHODS cific activity = 0.151 mCi/mg) and 21 dpm Samples to be dialyzed were placed in a nl-’ D-[ UJ4C]glucose (specific activity microchamber (devised by Stansbury) made = 0.049 mCi/mg) in glass-distilled water and from a 1.5-ml polyethylene microcentrifuge dispensed loo-p.1 aliquots into five microtube as follows (Fig. 1). First, a heated wire chambers. The microchambers were placed was used to melt a hole in the center of on a common dialysis wick and dialyzed the cap of the tube. Next, the tube was cut against glass-distilled water at 5°C. After with a razor as indicated, a square of dialysis various periods of time, one or two of the membrane was placed over the opening of microchambers were removed from the the cap, and the cap was pressed in place dialysis wick, and 55-nl aliquots (2) taken to seal the dialysis membrane between the for assay of 3H and 14C concentration. The lip and the cap. Care must be taken during remaining fluid in the dialysis chamber was this procedure to insure that the dialysis collected as completely as possible with a membrane is not damaged by excessive Pasteur pipet drawn to a fine tip, and weighed. A technique for dialysis of small samples (5-500 ~1) was recently described by Awdeh (1). In attempting to apply this technique to dialyze loo-p1 aqueous samples of [3H]inulin, we found that when the sample droplets were applied to the dialysis membrane as described by Awdeh (see his Fig. l), the droplets spread out, making recovery of the fluid impossible. We therefore developed the method described herein to allow containment and complete recovery of microliter samples after dialysis against large volumes of buffer. The principle is similar to that of Awdeh’s technique, except for placement of the sample to be dialyzed in a microchamber.
109
0003-2697/79/050109-03$02.00/0 Copyright B 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
110
BRAND AND STANSBURY
'OUTCENTER
0. ADD SAMPLE, GREASED COVERSLIP
DdYSlS MEMBRANE
ok DIALYSIS WICN
FIG. 1. Construction and use of microdialysis chamber.
Tritium and ‘“C concentrations in the nanoliter samples were assayed by standard dual channel liquid scintillation techniques. Radioisotopes were obtained from New England Nuclear ([3H]inulin) and ICN ([‘“Cl glucose). RESULTS
The concentration of 3H and 14C in the nanoliter samples taken from the microchambers dialyzed for different times are shown in Table 1, as well as the percentage recovery of dialysis fluid (based on the weight of fluid recovered, 100% = 0.100 g). Another similar experiment gave essentially identical results. TABLE
1
RECOVERY OF[W]INULIN AND [YJGLUCOSE AFTERDIALYSIS IN MICROCHAMBERS Dialysis time 0) 0 I8 24 48
dpm nl-’ RWWXy” no
[3HlInulin
2 2 I
42 23.8, 21.8 21.0, 22.0 21.2
a Number ofmicrocbambcn D Based on weight
[‘4C]Glucose
(%I
21 0.24, 0.25 0.20, 0.23 0.28
100.0, 102.5 102, 100.0 105.4
assayed.
of fIuid harvested
as described
in text.
DISCUSSION
From the data presented in Table 1, it may be concluded that complete separation of t3H]inulin and [14C]glucose was achieved within 18 h and that apparently complete recovery of volume may be achieved. Some comment on volume recovery is necessary, since during dialysis it is possible that backflow of solvent could occur, increasing the volume of fluid in the dialysis cell. From our data, it is unlikely that a large or continuous backflow of solvent occurred, since there was precise recovery of volume and the recovery of volume was constant with time from 18 to 48 h. Furthermore, if backflow of solvent were to occur, the driving force would be the difference in osmolality between the dialysand (distilled water) and the dialysate (containing [3H]inulin and [14C]glucose). In these experiments, we calculated from the manufacturer’s stated specific activity that the concentration of inulin and glucose, at the radioactive levels used, was - 1 rnosm . This low solute concentration in the dialysis cell would provide very little force for backflow of solvent. Therefore, while we cannot completely rule. out backflow of solvent affecting volume recovery, it is unlikely that such a backflow makes
IMPROVED
MICRODIALYSIS
a significant contribution to the volume of fluid recovered from the dialysis cell under these experimental conditions. Of course, in experiments in which a greater difference of osmolality across the membrane existed, greater net solvent flow might occur. Assuming that we are able to recover almost all the fluid added to the dialysis cell, then the decrease of 50% in apparent [3H] inulin concentration with dialysis means a net loss of 50% of the 3H has occurred. The simplest explanation for this loss, especially since the 3H concentration was constant after 18 h of dialysis, is that the original [3H]inulin contained a significant amount of small molecular weight fragments that could pass through the membrane. Contamination of commercial radioactive inulin with small molecular weight fragments has been reported previously (3). Zeppezauer has also reported construction
TECHNIQUE
111
of a microdialysis cell (4). The cell described here is simpler than Zeppezauer’s and allows easier access for addition or removal of sample. These results demonstrate the utility of this inexpensive method for purification of small volume samples with quantitative recovery of volumes possible in the microliter range. ACKNOWLEDGMENT This work was supported by a grant from the National Institutes of Health.
REFERENCES 1. Awdeh, Z. L. (1976)Anal. Biochem. 71,601-603. Quinton, P. M. (1976) J. Appi. Physioi. 40, 260262. 3. Levi, G. (1969) Anal. Biochem. 32, 348-353. 4. Zeppezauer, M. (1971) in Methods in Enzymology (Jakoby, W. B., ed.), Vol. 22, pp. 253-266, Academic Press, New York. 2.
BIOCHEMISTRY
94,109- 111 (1979)
Improved
Microdialysis
Technique
PAUL H. BRANDANDRACHELSTANSBURY Department of Physiology, Medical College of Ohio, CS #10008~ Toledo, Ohio, 43699 Received September 13, 1978 A simple, inexpensive method is described for dialysis of microliter amounts of aqueous samples against large volumes of solution with complete recovery of the fluid dialyzed. An example is given of application of the method to separation of [“Hlinulin from a monosaccharide.
pressure. To use, the sample is added, the upper surface covered with a greased coverslip, and, to commence dialysis, the chamber is placed on the dialysis wick [equivalent to the glass-wool pad, Ref. (1, Fig. Id)]. Other differences from Awdeh’s method are that in order to retain inulin (M, - 5500) we selected a dialysis membrane rated to retain substances of molecular weight ~3500 (A. H. Thomas); for convenience, a soft filter-paper strip (cut from a thin-layer chromatographic saturation pad, A. H. Thomas) was substituted for the glass-wool pad. To test the efficacy of the microchamber in separating [3H]inulin from small molecular weight compounds, we prepared a solution of 42 dpm nl-’ [3H]methoxyinulin (speMETHODS cific activity = 0.151 mCi/mg) and 21 dpm Samples to be dialyzed were placed in a nl-’ D-[ UJ4C]glucose (specific activity microchamber (devised by Stansbury) made = 0.049 mCi/mg) in glass-distilled water and from a 1.5-ml polyethylene microcentrifuge dispensed loo-p.1 aliquots into five microtube as follows (Fig. 1). First, a heated wire chambers. The microchambers were placed was used to melt a hole in the center of on a common dialysis wick and dialyzed the cap of the tube. Next, the tube was cut against glass-distilled water at 5°C. After with a razor as indicated, a square of dialysis various periods of time, one or two of the membrane was placed over the opening of microchambers were removed from the the cap, and the cap was pressed in place dialysis wick, and 55-nl aliquots (2) taken to seal the dialysis membrane between the for assay of 3H and 14C concentration. The lip and the cap. Care must be taken during remaining fluid in the dialysis chamber was this procedure to insure that the dialysis collected as completely as possible with a membrane is not damaged by excessive Pasteur pipet drawn to a fine tip, and weighed. A technique for dialysis of small samples (5-500 ~1) was recently described by Awdeh (1). In attempting to apply this technique to dialyze loo-p1 aqueous samples of [3H]inulin, we found that when the sample droplets were applied to the dialysis membrane as described by Awdeh (see his Fig. l), the droplets spread out, making recovery of the fluid impossible. We therefore developed the method described herein to allow containment and complete recovery of microliter samples after dialysis against large volumes of buffer. The principle is similar to that of Awdeh’s technique, except for placement of the sample to be dialyzed in a microchamber.
109
0003-2697/79/050109-03$02.00/0 Copyright B 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
110
BRAND AND STANSBURY
'OUTCENTER
0. ADD SAMPLE, GREASED COVERSLIP
DdYSlS MEMBRANE
ok DIALYSIS WICN
FIG. 1. Construction and use of microdialysis chamber.
Tritium and ‘“C concentrations in the nanoliter samples were assayed by standard dual channel liquid scintillation techniques. Radioisotopes were obtained from New England Nuclear ([3H]inulin) and ICN ([‘“Cl glucose). RESULTS
The concentration of 3H and 14C in the nanoliter samples taken from the microchambers dialyzed for different times are shown in Table 1, as well as the percentage recovery of dialysis fluid (based on the weight of fluid recovered, 100% = 0.100 g). Another similar experiment gave essentially identical results. TABLE
1
RECOVERY OF[W]INULIN AND [YJGLUCOSE AFTERDIALYSIS IN MICROCHAMBERS Dialysis time 0) 0 I8 24 48
dpm nl-’ RWWXy” no
[3HlInulin
2 2 I
42 23.8, 21.8 21.0, 22.0 21.2
a Number ofmicrocbambcn D Based on weight
[‘4C]Glucose
(%I
21 0.24, 0.25 0.20, 0.23 0.28
100.0, 102.5 102, 100.0 105.4
assayed.
of fIuid harvested
as described
in text.
DISCUSSION
From the data presented in Table 1, it may be concluded that complete separation of t3H]inulin and [14C]glucose was achieved within 18 h and that apparently complete recovery of volume may be achieved. Some comment on volume recovery is necessary, since during dialysis it is possible that backflow of solvent could occur, increasing the volume of fluid in the dialysis cell. From our data, it is unlikely that a large or continuous backflow of solvent occurred, since there was precise recovery of volume and the recovery of volume was constant with time from 18 to 48 h. Furthermore, if backflow of solvent were to occur, the driving force would be the difference in osmolality between the dialysand (distilled water) and the dialysate (containing [3H]inulin and [14C]glucose). In these experiments, we calculated from the manufacturer’s stated specific activity that the concentration of inulin and glucose, at the radioactive levels used, was - 1 rnosm . This low solute concentration in the dialysis cell would provide very little force for backflow of solvent. Therefore, while we cannot completely rule. out backflow of solvent affecting volume recovery, it is unlikely that such a backflow makes
IMPROVED
MICRODIALYSIS
a significant contribution to the volume of fluid recovered from the dialysis cell under these experimental conditions. Of course, in experiments in which a greater difference of osmolality across the membrane existed, greater net solvent flow might occur. Assuming that we are able to recover almost all the fluid added to the dialysis cell, then the decrease of 50% in apparent [3H] inulin concentration with dialysis means a net loss of 50% of the 3H has occurred. The simplest explanation for this loss, especially since the 3H concentration was constant after 18 h of dialysis, is that the original [3H]inulin contained a significant amount of small molecular weight fragments that could pass through the membrane. Contamination of commercial radioactive inulin with small molecular weight fragments has been reported previously (3). Zeppezauer has also reported construction
TECHNIQUE
111
of a microdialysis cell (4). The cell described here is simpler than Zeppezauer’s and allows easier access for addition or removal of sample. These results demonstrate the utility of this inexpensive method for purification of small volume samples with quantitative recovery of volumes possible in the microliter range. ACKNOWLEDGMENT This work was supported by a grant from the National Institutes of Health.
REFERENCES 1. Awdeh, Z. L. (1976)Anal. Biochem. 71,601-603. Quinton, P. M. (1976) J. Appi. Physioi. 40, 260262. 3. Levi, G. (1969) Anal. Biochem. 32, 348-353. 4. Zeppezauer, M. (1971) in Methods in Enzymology (Jakoby, W. B., ed.), Vol. 22, pp. 253-266, Academic Press, New York. 2.
