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A convenient technique for dialysis
180
SHORT
CO;LI1lUNIC!ATIONS
REFERENCES 1. COONS, A. H., (1942). 2. COONS, A. H., 3. RIOGS, J. L., T. G., Am. 4. HIRAMOTO, R., 611 (1958).
CREECH,
H. J., JONES,
R. N., AND BERLINER,
AND CAPLAN, M. H., J. Exptl. SEIWALD, R. J., BURCKHALTER, J. Pathol. 34, 1031 (1958). ENCEL, R., AND PRESSMANN,
E., J. Immunol.
Med. 91, 1 (1950). J. H., DOWNS, C. M., D.,
Proc.
sot.
Exptl.
45, 159
AND METCALF, Viol.
Med.
97,
LLOYD C. FELTOH C. ROBERT MCMILLION Hynson, Westcott d% Dunning, Inc. Raltimore, Maryland Received December IS, 1960
A ,Convenient
Technique
for Dialysis
In the course of studying dialyzable components in digests of connective tissue,l a method of dialysis was sought which would offer the following advantages over the conventional procedure of equilibrating the filled dialysis sac with a large volume of fluid: (a) nearly complet,e removal of dialyzable material, (b) a relatively small volume of dialyzate, and (c) a rapid rate of dialysis. The following procedure has been found to fulfill the foregoing requirements to a satisfactory degree. The filled sac is suspended from a support, and the dialyzing fluid is permitted to flow on the sac at a rate of approximately 2 ml/min. A small diameter cord is then wrapped around the bag in a spiral with only a slight pitch and with successive windings approximately 3 mm apart (Fig. In). The cord must wet readily so that surface tension of the fluid will hold it to the bag.2 When the sac has been wrapped, the flow of dialyzing fluid is adjusted to a slower rat,e to hold the dialyzate volume to a minimum. The data presented in Table 1 will serve to illustrate the effectiveness of the method described. The results were obtained by suspending over a fraction collector a Visking 18/32 sac, wrapped as described, containing 50 ml of 0.9 M ammonium sulfate and allowing water to flow down the l Connective tissue investigations aided by grants from Kansas Tuberculosis and Health Association, Jane Coffi Childs Memorial Fund for Medical Research, and U. S. Public Health Service (RG-6290). ’ Clark’s No. 8 Six Cord, not mercerized, is being used, but any similar cord would presumably be satisfactory.
SHORT
181
CONMUNICATIONS
Large Diameter Glass Tubing
Buffer
FIG. 1. Diagram of setup for dialysis. For dialysis against water the bag may be suspended in air (a); but for dialysis against buffers provision must be made to prevent changes in buffer concentration due to evaporation. This is accomplished by the arrangement shown in (5).
bag at a rate averaging 1.6 mi/min. The dialysis was carried out at room temperature. Fractions were collected at lo-min intervals. Aliquots of the dialyzate fractions were then analyzed for ammonia. It can be seen that 99% of the ammonia is removed in slightly less than 7 hr with a dialyzate volume only 13 times that of the solution dialyzed. These results were obtained without stirring the contents of the bag. Stirring would be expect.ed to increase the rate of dialysis (1) and would be particularly useful for large diameter tubing. The conditions given for dialysis of ammonium sulfate are not necessarily the most favorable from the standpoint of rate of flow of dialyzing TABLE REMOVAL
OF AMMONIA Time (min)
OS DIALYSIS removed
0 10 20 30 40 50 60 400b a Present in the bag: 89.4 mmoles. b Total volume of dialyzate: 647 ml.
1 OF
NH2 (mm&s)
0” 9.2 17.7 25.2 32.2 38.4 43.9 88.6
50 ML OF 0.9 M (N&)&O, NHr removed
0 10.3 19.8 28.2 36.0 42.9 49.1 99.2
(70 )
182
SHORT COMMUNICATIONS
fluid and minimum volume required. The data given serve simply to show the advantages of this technique compared to the conventional equilibration procedure. We routinely dialyze 100-ml volumes for 17-20 hr at 4OC with a flow of dialyzing fluid over the bag of about 50 ml/hr. This has served to reduce 1 M NaCl and 0.1 M phosphate buffer to less than 1O-4M solutions with about 10 vol of dialyzing fluid. The equilibration procedure would require 3-5 changes of 10 vol each. The procedure outlined permits almost complete recovery of a dialyzable substance in a minimum volume of solution. REFERENCES 1. OGSTON, A. G., Arch. B&hem.
Biophys. 89,191 (1900). VERNE D. HOSPELHOBN"
Lerrigo Memorial Laboratory University of Kansas Medical Center Kansas City, Kansas Received December 2’7, 1960 3 Fellow of the Helen Hay Whitney
Separation
Foundation
of Urinary 17-Ketosteroids Gas Chromatography
by
The principal 17-ketosteroids excreted by humans are androstan-3a-ol17-one (androsterone), 5/3-androstan-3a-ol-l7-one (5/3-androsterone, etiochoIanoIone) and A5-androsten-3,&ol-17-one (dehydroepiandrosterone) . These steroids are present in normal urine as suIfate and glucuronide conjugates, and their identification and estimation is of interest in studies of several endocrine abnormalities and in steroid metabolic studies. The free steroids may be obtained by solvent extraction after acid or enzymic hydrolysis of the conjugated excretion products,1 and the determination of total 17-ketosteroids is usually carried out by a calorimetric method based on the Zimmermann reaction (2), The recent development of gas chromatographic techniques for steroid ’ Variations in the hydrolysis procedure see, for example, the enzymic procedure tions. The hydrolysis problem is under J. A..-Cooper, Methodist Hospital, Texas
may atlect the yield of individual steroids; of Givner et al. (1) for estrogen determinainvestigation by Dr. B. G. Creech and Dr. Medical Center, Houston, Texas.
SHORT
CO;LI1lUNIC!ATIONS
REFERENCES 1. COONS, A. H., (1942). 2. COONS, A. H., 3. RIOGS, J. L., T. G., Am. 4. HIRAMOTO, R., 611 (1958).
CREECH,
H. J., JONES,
R. N., AND BERLINER,
AND CAPLAN, M. H., J. Exptl. SEIWALD, R. J., BURCKHALTER, J. Pathol. 34, 1031 (1958). ENCEL, R., AND PRESSMANN,
E., J. Immunol.
Med. 91, 1 (1950). J. H., DOWNS, C. M., D.,
Proc.
sot.
Exptl.
45, 159
AND METCALF, Viol.
Med.
97,
LLOYD C. FELTOH C. ROBERT MCMILLION Hynson, Westcott d% Dunning, Inc. Raltimore, Maryland Received December IS, 1960
A ,Convenient
Technique
for Dialysis
In the course of studying dialyzable components in digests of connective tissue,l a method of dialysis was sought which would offer the following advantages over the conventional procedure of equilibrating the filled dialysis sac with a large volume of fluid: (a) nearly complet,e removal of dialyzable material, (b) a relatively small volume of dialyzate, and (c) a rapid rate of dialysis. The following procedure has been found to fulfill the foregoing requirements to a satisfactory degree. The filled sac is suspended from a support, and the dialyzing fluid is permitted to flow on the sac at a rate of approximately 2 ml/min. A small diameter cord is then wrapped around the bag in a spiral with only a slight pitch and with successive windings approximately 3 mm apart (Fig. In). The cord must wet readily so that surface tension of the fluid will hold it to the bag.2 When the sac has been wrapped, the flow of dialyzing fluid is adjusted to a slower rat,e to hold the dialyzate volume to a minimum. The data presented in Table 1 will serve to illustrate the effectiveness of the method described. The results were obtained by suspending over a fraction collector a Visking 18/32 sac, wrapped as described, containing 50 ml of 0.9 M ammonium sulfate and allowing water to flow down the l Connective tissue investigations aided by grants from Kansas Tuberculosis and Health Association, Jane Coffi Childs Memorial Fund for Medical Research, and U. S. Public Health Service (RG-6290). ’ Clark’s No. 8 Six Cord, not mercerized, is being used, but any similar cord would presumably be satisfactory.
SHORT
181
CONMUNICATIONS
Large Diameter Glass Tubing
Buffer
FIG. 1. Diagram of setup for dialysis. For dialysis against water the bag may be suspended in air (a); but for dialysis against buffers provision must be made to prevent changes in buffer concentration due to evaporation. This is accomplished by the arrangement shown in (5).
bag at a rate averaging 1.6 mi/min. The dialysis was carried out at room temperature. Fractions were collected at lo-min intervals. Aliquots of the dialyzate fractions were then analyzed for ammonia. It can be seen that 99% of the ammonia is removed in slightly less than 7 hr with a dialyzate volume only 13 times that of the solution dialyzed. These results were obtained without stirring the contents of the bag. Stirring would be expect.ed to increase the rate of dialysis (1) and would be particularly useful for large diameter tubing. The conditions given for dialysis of ammonium sulfate are not necessarily the most favorable from the standpoint of rate of flow of dialyzing TABLE REMOVAL
OF AMMONIA Time (min)
OS DIALYSIS removed
0 10 20 30 40 50 60 400b a Present in the bag: 89.4 mmoles. b Total volume of dialyzate: 647 ml.
1 OF
NH2 (mm&s)
0” 9.2 17.7 25.2 32.2 38.4 43.9 88.6
50 ML OF 0.9 M (N&)&O, NHr removed
0 10.3 19.8 28.2 36.0 42.9 49.1 99.2
(70 )
182
SHORT COMMUNICATIONS
fluid and minimum volume required. The data given serve simply to show the advantages of this technique compared to the conventional equilibration procedure. We routinely dialyze 100-ml volumes for 17-20 hr at 4OC with a flow of dialyzing fluid over the bag of about 50 ml/hr. This has served to reduce 1 M NaCl and 0.1 M phosphate buffer to less than 1O-4M solutions with about 10 vol of dialyzing fluid. The equilibration procedure would require 3-5 changes of 10 vol each. The procedure outlined permits almost complete recovery of a dialyzable substance in a minimum volume of solution. REFERENCES 1. OGSTON, A. G., Arch. B&hem.
Biophys. 89,191 (1900). VERNE D. HOSPELHOBN"
Lerrigo Memorial Laboratory University of Kansas Medical Center Kansas City, Kansas Received December 2’7, 1960 3 Fellow of the Helen Hay Whitney
Separation
Foundation
of Urinary 17-Ketosteroids Gas Chromatography
by
The principal 17-ketosteroids excreted by humans are androstan-3a-ol17-one (androsterone), 5/3-androstan-3a-ol-l7-one (5/3-androsterone, etiochoIanoIone) and A5-androsten-3,&ol-17-one (dehydroepiandrosterone) . These steroids are present in normal urine as suIfate and glucuronide conjugates, and their identification and estimation is of interest in studies of several endocrine abnormalities and in steroid metabolic studies. The free steroids may be obtained by solvent extraction after acid or enzymic hydrolysis of the conjugated excretion products,1 and the determination of total 17-ketosteroids is usually carried out by a calorimetric method based on the Zimmermann reaction (2), The recent development of gas chromatographic techniques for steroid ’ Variations in the hydrolysis procedure see, for example, the enzymic procedure tions. The hydrolysis problem is under J. A..-Cooper, Methodist Hospital, Texas
may atlect the yield of individual steroids; of Givner et al. (1) for estrogen determinainvestigation by Dr. B. G. Creech and Dr. Medical Center, Houston, Texas.