Thin-Film Dialysis of Glucose Reynold John, Augustine Appiah, and Lawrence M. Marshall D e p a r t m e n t of B i o c h e m i s t r y , College of M e d i c i n e , H o w a r d University, Washington, D. C. 20001
During countercurrent distribution ( I ) , differences in mobility and therefore differences in the distribution coefficients were observed between [l-l4C]arabinose and its unlabeled counterpart. When the label was on carbon 5 instead of carbon 1. there was no isotope effect. When methyl glycosides were prepared ( Z ) , no isotope effect was discernible during countercurrent distribution, but when the glycosides were hydrolyzed, the isotope effect for the sugar could be observed again. It occurred to us that carbon-14 on carbon 1 possibly influenced the conformation of the pentose or possibly the equilibrium constant of the anomerization or both with the consequence that the distribution coefficient of the sugar was changed. Since analytical dialysis rates are known to be influenced by any condition that will alter the long cross section of a solute. and since conformation and firm solvation can be two such influences. simple dialysis of labeled glucose was examined in an attempt to gain additional insight into positional isotope effects. At the time the study was begun we had in mind reports ( 3 ) of Craig that suggested that his observed effects of solute heterogeneity on the dialysis rates of sugars might be related to anomeric equilibria. Accordingly. we measured the relative rates of escape of the anomers of glucose. Although our data failed to unequivocally establish a relationship between isotope effects and a-p isomerism of the sugar, the results are reported here because, insofar as we know. no similar observations during dialysis have been reported elsewhere.
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of t h e dialysate of o - g l u c o s e originally in a n o m e r i c e q u i l i b r i u m (A) Escape at 15 min. (B) Escape at 75 min. As integrated by the paper weight method, the ratio of areas corresponding to the a to d anomer is 0.493. Inset: original sample before dialysis, area equivalent to the ct to l3 anomer i s 0.58. The retention times of the a and 3 anomers are 3.5 and 5.5 min. The carrier gas was helium
EXPERIMENTAL Dialysis casings KO, 10886 from R i l l Corporation were 19 mm when round. ~ - [ l - ~ * C ] G l u c o specific se. activity 4.5 mCi,/mM and ~ - [ 6 - ~ * c ] g l u c o sspecific e, activity 3.3 and 5.0 mCi,/mM were obtained from New England Nuclear Corp. 8-D-Glucose was purchased from K & K Laboratories. Reagents for silylation were obtained from Fisher Scientific Co. Anomeric sugars were chromatographed on a Beckman G.C. 45 in accordance with the procedure of Bentley and Botlick ( 3 ) . Five microliters of sample were placed in 0.1 ml of dimethylformamide, rapidly mixed, and rapidly frozen in liquid nitrogen. One milliliter of silylating reagent was added on top of the frozen mass and the entire mixture was vigorously stirred over a 30-min period. Portions varying between 0.2 to 1.0 p1 of the solution were injected into a column of 4 ft X 94 in. Chromosorb FV. 108-120 mesh, containing 3% SE-30. The earlier (j)procedure of Craig was used for the dialyses. and membranes were acetylated by the method of Craig and Konigsberg (6). The area of the membrane exposed to dialysis was 90 mm2, Radioactivity was measured in a Suclear Liquid Scintillation System, Model 8264. Dioxane was the solvent for the counting solution that contained per liter 70 g of naphthalene. 7 g of 2,5-diphenyloxazole. and 0.25 g of p-bis[2-(5-phenyloxazolyl)benzene]. As the index of chemical measurement, glucose was measured by ferricyanide reduction in the Technicon AutoAnaIyzer. (1) L. M. Marshali and R . E. Cook, J. Amer. Chem. Soc., 84, 2647
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(1962). L. M. Marshall, W. P. Walker, J. A. Gunn, and L. Panton, J. Chromafogr., 29, 103 (1967) K . K . Stewart, L. C. Craig, and R. C. Williams, Jr., Anal. Chem., 42. 1252 (1970). R . Bentley and N . Botlick, Anal. Biochem., 20, 312 (1967) L. C. Craig and A. 0. Pulley, Biochemistry, 1, 89 (1962). L. C. Craig and W. Konigsberg, J. Phys. Chem., 65, 166 (1961).
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Figure 2. G a s c h r o m a t o g r a m s f r o m r e t e n t a t e s a m p l e s o f D-gluc o s e in a n o m e r i c e q u i l i b r i u m b e f o r e dialysis (A) After 60-min dialysis through an unacetyiated membrane As integrated by the paper weight method areas representing the ct to d anomers are in the ratio of 0 8 (B) After 2-hr dialysis through an acetyiated membrane Similarly integrated areas representing the ct to d anomer are in the ratio of 1 1 The retention times of the ct and 3 anomers are 3 5 and 5 5
ANALYTICAL CHEMISTRY. VOL. 4 5 , NO. 1 2 , OCTOBER 1973
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Figure 4. Escape curves for [l-14C]glucose ( X ) , [6-14C]glucose and D-glucose (0)
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Figure 3. Escape curves for [l-i4C]glucose ( X ) , [6-14C]glucose and unlabeled D-arabinose ( 0 )
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Samples were dialyzed in H20 through cellophane that had not been acetylated. Each isotope separately was dialyzed with the pentose, and since the two plots of the pentose coincided, the dialysis of the pentose is shown as a single line. Five microcuries in 5 ml of H20 of each labeled sugar was used. Escape rates, expressed as the slopes of the above plots, for [6-14C]glucose, unlabeled D-arabinose, and [l-14C]glucose are (a) before 6 0 min, 6.3 X 7.0 X and 9.3 X (b) after 60 rnin, 4.6 X 6.4 X and 6.4 X
Samples were dialyzed in 0.1M acetate buffer, pH 5.5. through cellophane that had been acetylated. Each isotope separately was dialyzed with the hexose and since the two plots of the escape rates for the hexose coincided, the dialysis of the hexose is shown a s a single line. Five microcuries in 5 ml of acetate buffer of each labeled sugar was used. Escape rates expressed as in Figure 3 for D-glucose, [6-14C]glucose. and [l-'4C]glucoseare 1.8 X 2.2 X and 2.7 X
expected, escaped more rapidly than the slower diffusing [6-14C]glucose. With acetylated membranes (Figure 4 ) , similar relative rates for the positionally labeled sugars To test the radiochemical purity of ~ - [ l - ~ ~ C ] g l u cand o s e ~ - [ 6 - were observed but glucose labeled on either carbon 6 or carbon 1 escaped more rapidly than its unlabeled counter14C]glucose, each was mixed with 20 pCi of ~ - [ 6 - ~ H ] g l u c oand se part. The data show that there is a positional isotope ef10 mg of unlabeled glucose and incubated with 20 mg of glucose oxidase (Schwarz Bioresearch, 200 units/mg) a t 25 "C in 0.1M fect during dialysis. When the 30-min dialysate sample acetate buffer, p H 5.5 in a 5-ml volume. The reaction was was divided into two parts and dialyzed again. before and stopped by boiling, and 0.5 to 1 ml of the final mixture was chroafter standing 24 hr to reach anomeric equilibrium, the matographed on Dowex 1-X8 columns, 17 cm X 18 m m . One hundata. Figure 5 , indicate that the diffusate sample underdred and forty milliliters caused the glucose to emerge. Gluconowent mutarotation. lactone followed when the influent mixture gradually increased Positional isotope effects involving sugars during counwith respect to 0.5Ai formic acid concentration. Tritium and carbon-14 were counted simultaneously with the spectrometer winterpart distribution have been reported for arabinose ( I ) and dows set so t h a t tritium was counted with a n efficiency of 26% in lyxose ( 7 ) . In the account of these investigators, it was one channel and carbon with an efficiency of 40% in the other. At pointed out that such effects are reminiscent of isotope the end of 20 min, there was no change in carbon-14:tritium raeffects that diminished as the position of the radioactive tios for ~-[6-~*C]glucose or ~ - [ l - ~ ~ C ] g l u c in o s eeither the product carbon was farther in the molecule from the carboxyl or the residual glucose, and the specific activity of the glucose, group when amino acids were chromatographed on Dowex both forms, expressed as cts/min/mg glucose was the same in the starting and residual glucose. (8). Piez and Eagle ( 8 ) believed that the difference in electronegativity between carbon-14 and carbon- 12 acRESULTS AND DISCUSSION counted for the amino acid data. If such reasoning were Heterogeneity of the solute is evident from the gas chroapplicable to the isotope effect observed during dialysis, matogram, Figure 1. In the first 15 min, the (3 escapes one would rationalize structural modification of the conmore rapidly than the cy anomer and a t 7 5 min, the correformers of the hexose as a consequence of the influence of sponding ratio (0.49) approaches but does not reach that the isotope on nonbonded interactions as well as the interof the original solute (0.58). That the retentate is enriched actions between the C1 hydroxyl and the ring oxygen of' with respect to the cy anomer is shown in Figure 2 . the sugar. Such an isotope effect could not only influence ~ - [ l - ~ ~ C ] G l u c oescaped se more rapidly than ~ - [ 6 14C]glucose and even unlabeled arabinose in unacetylated (7) L. M. Marshall and D. Magee. J. Chromatogr.. 15, 97 (1964). membranes (Figure 3). Unlabeled arabinose, as would be (8) K . A . Piez and H.Eagle, J. Arner. Chem. Soc.. 78, 5284 (1956) ANALYTICAL CHEMISTRY, VOL. 45, NO. 12, OCTOBER 1973
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the mutarotation equilibrium but-to the extent that it controls the length of the long cross section of a conformer-could narrow the distribution of the conformer population of the labeled solute in a manner that would enhance the dialysis rate. Such control could account for the isotope effect during dialysis being greater than that observed during countercurrent distribution ( I , 2, 7 ) . An alternate explanation could be advanced if the control extended to the hydration of the conformer or hydration and length of the long axis of it. Such control would relate isotope effects during dialysis to the rate enhancements in reactions (9) that result from structural modifications caused by improvement in the angular relation of nucleophilic and electrophilic centers and exclusion of solvent between reaction partners as well as their desolvation. It would then seem that the inclusion of considerably larger rate changes in theories for kinetic isotope effects than those assumed for molecules that do not change their conformation is mandatory. The difference in the dialysis rates of the a and p anomers can have implications in long term dialysis techniques to correct chemical changes ( I O ) through hemodialysis, because any anomeric imbalance could influence activity of enzymes that are specific for one or the other anomer. Enzymes that catalyze reactions involving carbon 1 of phosphate esters of glucose are known to have such specificity ( 1 1 ) .
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Received for review February 16, 1973. Accepted April 13, 1973. This research was supported in part by General Research Support Grant 5Sol-RR05361-10 of the United States Public Health Service and a grant from E. I. Du Pont de Nemours and Company.
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The conditions for the dialysis were the same as those for Figure 3 except t h a t t h e 30-min d i a l y s a t e was d i v i d e d i n t o parts, One).-.( was dialyzed at once the other ( B - B ) after anomeric equilibrium. Escape rates, expressed as in Figure 3, for the sample at once and after equilibrium, respectively, are (a) before 60 min, 8.1 X and 6.2 X ( b ) after 60 min, 7.1 X and 4.5 X
(9) Such enhancement is called stereopopulation control by Cohen: S. Milstien and L. A . Cohen, J. Amer. Chern. SOC., 94, 9158 (1972); R . T . Borchardt and L. A . Cohen, ibid., 94, 9166 (1972);ibid., 94, 9175 - - (19771 (lo) P. E, Teschan, Amer. J. Med.. 48,671 (1970). (11 ) M . Salas, E. Vinuela, and A . Sols, J. Bioi. Chern., 240,561 (1965). \
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High Speed, High Pressure Liquid Chromatography of Similar Tropane Alkaloids Martin H. Stutz and Samuel Sass Analytical Chemistry Branch, Chemical Research Division. Chemical Laboratory, Edge wood Arsenal, Aberdeen Proving Ground, Md
21010
Liquid chromatography as a separate analytical technique has seen a rebirth during the past few years. A good summary Of Some Of the recent effort can be found in the compilation by Kirkland (11, and through a Perusal o f t h e recent literature (2-17). This paper deals with the application of high speed liquid chromatography to the separation and quantitative determination of some physically active compounds of similar structure. ~t demonstrates the applicability and usefulness of liquid chromatographS for differentiating between compounds which might have similar chemical structure but differing physiological effects. 2134
EXPERIMENTAL Apparatus. All experiments were performed on a ?;ester-Faust Model 1200 Liquid Chromatograph (Perkin-Elmer, Norwalk, Conn.). The chromatograph was equipped with dual high pressure (2000 psi) pumps and a n automatic solvent transport system which controls selection of pumps and eliminates the need to make changes in system plumbing when changing modes of ~ e r ation (i e , from step to gradiant elution). It also makes it possible to pre-program an elution profile and have the analysis carried out automaticallv. Sample application to the column was made with a ~ 5 - Hamilton ~ 1 syringe( ~ ~ company, ~ i whittier, l ~ ~ Calif.) through a septum injector directly above the column, A 4.6-mm (i.d.) X 1-meter stainless steel column was dry packed
A N A L Y T I C A L C H E M I S T R Y , VOL. 45, NO. 1 2 , OCTOBER 1973
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