Rapid Determination of [Carbon-14] Glucose Specific Radioactivity for In Vivo Glucose Kinetics ~ S. P. SCHMIDT, J. A. SMITH, and J. W. YOUNG Department of Animal Science Iowa State University Ames 50010

gives kinetic results similar to those calculated from the glucose-pentaacetate method.

ABSTRACT

Specific radioactivity of blood glucose was determined by use of a Dowex-1 anion-exchange column after [carbon-14] glucose was infused for in vivo kinetics. The first 20-ml eluate of protein-free blood filtrate from the column was discarded; then 10 ml was collected, lyophilized, and counted in a liquid-scintillation counter. Glucose concentration was determined and specific radioactivity calculated. Kinetic results were comparable to those obtained with the more laborious glucose-pentaacetate procedure.

MATERIALS

Anion-exchange resin was Dowex 1-X8, 50--100 mesh and Dowex l-X8, 1 0 0 - 2 0 0 mesh. The chloride and acetate forms work equally well. Wash the resins in deionized water and decant the fines. Glass columns were designed for a 1-cm x 16-cm resin bed and with a reservoir so blood filtrate can be loaded on the resin bed for continuous flow. XDC liquid scintillation counting solution (2) was 1 part xylene; 3 parts dioxane; 3 parts 2-ethoxyethanol; 1.0% 2,5-diphenyloxazole (PPO); .05% 1,4-bis-2-(5-phenyloxazolyl)-benzene (POPOP); 8.0% naphthalene. Other scintillation solutions that give acceptable counting efficiencies in the presence of water can be used.

INTRODUCTION

Kinetic studies of in vivo glucose metabolism by isotope-dilution techniques require separation of glucose from interfering labeled metabolites in the blood. Jones (3) has developed a procedure for converting glucose to the pentaacetate derivative which can be isolated easily and is soluble in toluene-based, liquid-scintillation solutions. The procedure has been used widely in recent experiments involving glucose kinetics (1, 4, 5, 6, 8, 9, 10) because it is highly repeatable, relatively simple, and apparently specific for glucose. However, because several steps are involved in forming, purifying, and isolating the pentaacetate derivative, the procedure is time consuming. Thus, it was desirable to develop a procedure that would give high reliability but require much less time. This report describes a rapid method for determining specific radioactivity of blood glucose which

PROCEDURE

Received March 22, 1974. 1Journal Paper No. J-7735 of the Iowa Agriculture and Home Economics Experiment Station, Ames. Project 1910. The work was supported in part by funds provided by Grant AM-10706, U.S. Department of Health, Education and Welfare.

Protein-free filtrates from 5 to 10 ml blood are obtained after a 1:10 dilution by the method of Somogyi (7). This yields a pH-7 filtrate, which is necessary for separation of low-pKa organic acids from glucose. Using distilled water, prepare a series of 1-cm x 16-cm columns after mixing washed Dowex resins in the ratio of 4 parts 50 to 100 mesh to 1 part 100 to 200 mesh. This resin ratio is a convenient way to adjust flow rates to approximately 1 ml/min. Conventional methods of controlling flow rate would have required careful cleaning of more glassware and equipment to avoid cross-contamination of radioactivity between successive samples. Allow protein-free blood filtrate to flow continuously through the column. Discard the first 20 ml of eluate, then collect and pipette 10 ml of eluate into a counting vial. Lyophilize or air-dry the sample in the vial. Dissolve the

952

TECHNICAL NOTE

dry powder in 1 ml water, add 15 ml XDC counting solution (2), and count in a liquidscintillation counter. The glucose content of the eluate is determined by the glucose oxidase method. 2 Specific radioactivity of glucose can be calculated by conventional methods. The columns were flushed with about 100 ml water between blood filtrates and used repeatedly for six samples before replacing the ion-exchange resin. An elevated reservoir increased the water flow rate to approximately 6 ml/min, shortening the time required between samples. This general procedure makes it possible to run several columns and samples simultaneously without having to recharge the resin bed between filtrates. The blood filtrates used for comparison were from nine glucose-kinetic experiments.3 In these experiments, steers averaging 185 kg were given 100 /aCi of [U-14C]glucose by single injection into the jugular vein, and 20 blood samples were taken over 4 h.

9 53

RESULTS AND, DISCUSSION

The data in Table 1 show that after 20 ml of blood filtrate have gone through the column, the glucose concentration in the eluate has returned to that of the original filtrate, and radioactivity has reached a plateau slightly less than in the original filtrate. This indicates radioactivity not in glucose has been removed. Thus, after discarding the first 20 ml of eluate, the 10-ml eluate collected for counting has the same glucose concentration as the original filtrate. Trapped radioactive organic acids can be washed from the resin with 1 M NaC1. The method separates glucose from lactate, an abundant metabolite of glucose metabolism, and from propionate, a major gluconeogenic precursor in ruminants (Table 2). When approximately 1 x 106 cpm in 60 /lmol of lactate were loaded on the column, only .22% of the cpm washed through with 50 ml of water. Then, 50 /~mol of [14C]glucose (2.7 x 105 cpm) in 2 ml water were added to the column and washed with 60 ml of water. Recovery of added glucose was 99.3% with 87% eluting in the first 10 ml. Similar results were obtained 2 Worthington Biochemical Corp., Freehold, NJ. with propionate. The amount of glucose added 3Trott, D. R., M.S. thesis, Iowa State University, to the column in these experiments (Table 2) Ames.

TABLE 1. Volume necessary to eliminate dilution of blood filtrate by void volume of column.

Radioactivity in eluate a

Glucose concentration in eluate b

(dpm/ml)

(m~100 ml)

478

6.70

1 2 3 4 5 6

195 406 436 449 453 453

2.95 6.17 6.48 6.64 6.62 6.60

1 2 3 4

46 41 24 13

0 0 0 0

Successive fractions (5 ml) Direct analyses of blood filtrate Analyses of blood filtrate eluate through ion exchange column

Analyses of 1 M NaCI eluate after washing column with H20

al ml protein-free blood filtrate or eluate was counted in 15 mI XDC (2). bBlood was diluted 1:10 during protein precipitation. Therefore, blood glucose concentration was 67 mg/lO0 ml. Journal of Dairy Science Vol. 58, No. 6

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SCHMIDT ET AL.

TABLE 2. Separation of lactate a or propionate b from glucose by Dowex-1.

Step in elution procedure

Successive fractions

Elution with water after lactate or propionate addition

(10 ml) 1 2 3 4 5

Elution with water after glucose addition

6 7 8 9 10 11

Wash with 1MNaCIto elu~ organic acids

12 13 14 15 16 17

Lactate radioactivity in eluate c

Propionate radioactivity in eluate c (cpm)

1,480 420 160 6O 90 2,210 253,760 11,020 2,830 160 160 150 268,080

109 7 22 0 24 162 230,252 23,173 1,564 235 28 41 255,293

781,920 87,450 19,250 2,930 1,350 700 893,600

322,281 126,753 4,781 711 245 124 454,895

aA 2.0 ml solution containing 1 × 106 cpm [14C]Na-lactate in 60 ~mol unlabeled lactate (pH 7.0) was pipetted on the column and eluted with water. Then, 2.7 × 105 cpm [14C]glucose in 50 ~mol unlabeled glucose in a 2.0 ml solution was loaded on the column and eluted with water followed by washing with 1 M NaCI. bA .5 ml solution containing 4.55 × 105 cpm [14C]Na-propionate in 60 ~mol unlabeled propionate (pH 7.0) was pipetted on the column and eluted with water. Then, 2.57 × 105 cpm [14C] glucose in 60 /1tool unlabeled glucose in a .5 ml solution was loaded on the column and eluted with water followed by washing with 1 M NaCI. CRecovery percentages can be determined by dividing total cpm in eluate by cpm in either lactate, propionate, or glucose loaded on the column times 100.

was approximately five times the amount used with blood filtrates diluted 1:10 by the Somogyi procedure (Table 1). Results equivalent to those in Table 2 also are obtained when unlabeled Somogyi filtrate is the eluting solvent instead of distilled water. Thus, anion-exchange resin can be used conveniently to separate blood glucose from organic acids having a tow pKa. Our method is valid for either studies of glucose kinetics or studies of gluconeogenesis from organic-acid precursors such as propiohate. To verify further the validity of the ionexchange method, the specific radioactivities of blood glucose from 154 blood samples obtained during nine in vivo glucose kinetic experiments were compared after being determined by both the established pentaacetate Journal of Dairy Science Vol. 58, No. 6

procedure of Jones (3) and the procedure described here. The correlation between values by the two methods was .996, and the regression coefficient was .964 (Fig. 1). Thus, the two methods gave similar results. Fig. 2 is a comparison of curves of glucose specific radioactivity vs. time from one experiment in which specific radioactivities were determined by forming the pentaacetate, by using the ionexchange column, or by counting the blood filtrate directly. Specific radioactivity values from either the pentaacetate method or the ion-exchange method gave similar results with considerable crossing of the two curves. The direct-count method gave a curve that was considerably higher (average 35%) than the others; however, the difference was not always this large. Thus, both the ion-exchange method

TECHNICAL NOTE

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FIG. 1. Correlation between glucose specific radioactivity values obtained by the glucose pentaacetate method and by the use of ion exchange resin t o remove contaminating organic acids.

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MINUTES

and the pentaacetate m e t h o d are r e m o v i n g radioactive contaminants. In the nine experiments in which the data were compared, values by either the p e n t a a c e t a t e or ion-exchange m e t h o d s were always similar. Further, we have tested our m e t h o d for b o t h 12 h single injection and 4 h c o n t i n u o u s infusion e x p e r i m e n t s and have obtained results equivalent to those described in detail in this paper. The critical test, however, is w h e t h e r kinetic measures calculated f r o m our m e t h o d are equivalent t o those calculated f r o m glucose specific radioactivities o b t a i n e d by the p e n t a a c e t a t e procedure. F r o m data typical of that shown in Fig. 2, glucose p o o l size, total e n t r y rate, and irreversible loss were calculated as described by

FIG. 2. Comparison of glucose specific radioactivity determined by three methods for studies of glucose kinetics after a single injection of 114C] glucose• (The direct counts were made by counting 1 ml blood filtrate in 15 ml XDC.)

White et al. (9) for six of the e x p e r i m e n t s (Table 3). Only six o f the nine sets of data were c o m p a r e d in Table 3 because the calves were fasted during t w o of the e x p e r i m e n t s and the ion-exchange data were i n c o m p l e t e for a third. There were no significant differences b e t w e e n the pentaacetate and ion-exchange m e t h o d s used to d e t e r m i n e glucose pool size, total e n t r y rate, and irreversible loss. These data indicate that characteristics of glucose kinetics deter-

TABLE 3. Comparison of glucose kinetic measures a calculated from glucose specific radioactivities determined by glucose pentaacetate and Dowex-1 anion exchange column.

Method

Pool size

Total entry rate

(g) Glucose pentaacetate Ion exchange column

28.7 +- 2.4 30.7 -+ 3.0

Irreversible loss (mg/min)

997 -+ 54 940 +- 52

472 -+ 30 465 -+ 37

aDetermined by resolving the 14C-glucose disappearance curve into two exponential components as described by White et al. (9). Values given as mean -+ SE (n=6). Means in the same column are not different (P > .10). Journal of Dairy Science Vol. 58, No. 6

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SCHMIDT ET AL.

mined by the m o r e rapid ion-exchange m e t h o d agree with those d e t e r m i n e d by the pentaacetate procedure of Jones (3). A f t e r b l o o d filtrates are obtained, specific activity values can be obtained by the ion-exchange m e t h o d in about o n e - f o u r t h the time required for the pentaacetate m e t h o d . The ion-exchange m e t h o d described here will not remove all amino acids that m i g h t b e c o m e labeled during [ 14 C] glucose infusions. Our results, however, indicate that labeled amino acids are n o t a major c o n t a m i n a n t of the blood filtrates. A m i n o acids could be r e m o v e d with t a n d e m columns e m p l o y i n g cation and anion exchange resins. Labeled bicarbonate w o u l d be f o r m e d during [ 14C] glucose metabolism. We have c o n d u c t e d studies, however, which show that added [t 4C] bicarbonate does n o t pass through the resin c o l u m n nor does it c o n t a m i n a t e glucose pentaacetates f o r m e d f r o m blood filtrates.

3.

4.

5.

6. 7. 8. 9.

REFERENCES

1. Argenzio, R. A., and H. F. Hintz. 1972. Effect of diet on glucose entry and oxidation rates in ponies. J. Nutr. 102:879. 2. Bruno, G. A., and J. E. Christian. 1961. Determin-

Journal of Dairy Science Vol. 58, No. 6

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ation of carbon-14 in aqueous bicarbonate solutions by liquid scintillation counting techniques. Anal. Chem. 33:1216. Jones, G. B. 1965. Determination of the specific activity of labeled blood glucose by liquid scintillation using glucose pentaacetate. Anal. Biochem. 12:249. Judson, G. J., E. Anderson, J. R. Luick, and R. A. Leng. 1968. The contribution of propionate to glucose synthesis in sheep given diets of different grain content. Brit. J. Nutr. 22:69. Judson, G. J., and R. A. Leng. 1972. Estimation of the total entry rate and resynthesis of glucose in sheep using glucoses uniformly labeled with 14C and variously labeled with 3H. Aust. J. Biol. Sci. 25:1313. Leng, R. A., J. W. Steel, and J. R. Luick. 1967. Contribution of propionate to glucose synthesis in sheep. Biochem. J. 103:785. Somogyi, M. J. 1945. The determination of blood sugar. J. Biol. Chem. 160:69. Ulyatt, M. J., F. G. Whitelaw, and F. G. Watson. 1970. The effect of diet on glucose entry rates in sheep. J. Agr. Sci. Camb. 75:565. White, R. G., J. W. Steel, R. A. Leng, and J. R. Luick. 1969. Evaluation of three isotope-dilution techniques for studying the kinetics of glucose metabolism in sheep. Biochem. J. 114:203. Wiltrout, D. W., and L. D. Satter. 1972. Contribution of propionate to glucose synthesis in the lactating and nonlactating cow. J. Dairy Sci. 55:307.