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Enzymatic assays for 2-deoxyglucose and 2-deoxyglucose 6-phosphate
ANALYTICAL
BIOCHEMISTRY
161,508-5 13 (I 987)
Enzymatic Assays for 2-Deoxyglucose
and 2-Deoxyglucose
6-Phosphate*,’
MAGGIE M.-Y. CHI, MARY ELLEN FLJSATERI,JOYCE G. CARTER, BEVERLY J. NORRIS, DAVID B. MCDOUGAL, JR., AND OLIVER H. LOWRY Departments
of Pharmacology School
and of Neurology and Neurosurgery, Washington of Medicine, St. Louis, Missouri 63110
University
Received November 3. I986 Methods for 2-deoxyglucose (2-M;) and 2-deoxyghtcose 6-phosphate (DG6P) are described which are based on the fact that DG6P is oxidized by glucose-6-phosphate dehydrogenase (G6PDH). but at a rate lOOO-fold slower than for ghrcose 6-phosphate, whereas hexokinase phosphorylates 2DG and glucose at comparable rates. Therefore, by adding the two enzymes in a suitable order, and in appropriate concentrations, 2DG. glucose, DG6P, and glucose 6-P can all be separately measured. To avoid a side reaction from the use of a high level of G6PDH, when measuring DG6P, glucose is first removed with glucose oxidase plus aldose reductase. 0 1987 Academic
Press, Inc.
2-deoxyglucose; 2-deoxyglucose 6-phosphate; glucose-6-phosphate dehydrogenase kinetics; enzymatic assay. KEY
WORDS:
2-Deoxyglucose (2DG)’ has become a very useful tool in the study of brain metabolism. Glucose and 2DG are similarly transported into brain and phosphorylated by hexokinase. However, 2-deoxyglucose 6-phosphate (DG6P) cannot be either converted to an analog of fructose 6-phosphate, or rapidly metabolized otherwise. Therefore, after 2DG administration, nearly all the DG6P that is formed accumulates, and this accumulation can provide a measure of glucose consumption. Because glucose is almost the exclusive fuel for brain, glucose consumption is a useful measure of brain energy metabolism and therefore of neuronal activity. In 1977 Sokoloff et al. described how to exploit this fact, with [14C]2DG, in a classic paper (1). This * This paper is dedicated to the memory of Nathan 0. Kaplan. ’ Supported by grants from the American Cancer Society (BC-4-29). and the U.S. PHS (NS-08862. NS-18387, NS-20762). * Abbreviations used: 2DG, 2-deoxyghrcose; DG6P. 2-deoxyglucose 6-phosphate; G6P, glucose 6-phosphate; G6PDH, glucose-6-phosphate dehydrogenase; 6PGDH, 6-phosphogluconate dehydrogenase; BSA. bovine serum albumin: PTZ, pentylenetetrazole. 0003-2697187 $3.00 Copyright Q 1987 by Academic Press, Inc. Al! rights of reproduction in any form reserved.
508
now widely used radioautographic method requires a 30- to 45-min time lapse after 2DG injection for dissipation of most of the unphosphorylated 2DG. The time lag limits the method to the study of events which occur on a long time scale. This report describes sensitive enzymatic methods for measuring both 2DG and 2DG6P as well as glucose and glucose 6phosphate (G6P) in the same tissue sample. With these methods, it has proven possible to measure changes in brain glucose consumption on a time scale of 1 to 5 min. The methods are based on the fact that both G6P and DG6P can be oxidized by G6P dehydrogenase (G6PDH) but at rates that differ by a factor of more than a thousand. Therefore, the NADH or NADPH produced with a low level of enzyme is a measure of G6P, while that produced with a much higher G6PDH level is DG6P. Glucose and 2DG are similarly distinguished after phosphorylation by hexokinase. For higher levels of these compounds (0.5 nmol or more) the fluorescence of the NADPH generated is measured directly. For lower levels (down to a few femtomoles if
ENZYMATIC
ASSAYS
FOR
2-DG
AND
DG6P
509
necessary), the NADPH is amplified by en- Direct Assays (0.5 to IO nmol in Each Aliquot) zymatic cycling (2,3). Two problems were encountered, both resulting from the use of such a high level of The analysis of a solution such as a tissue G6PDH. Commercial preparations of extract which contains all four components, G6PDH almost invariably contained traces 2DG, 2DG6P, glucose, and G6P, is made in of 6-phosphogluconate dehydrogenase. This two parts. caused errors in DG and DG6P readings Part A. An aliquot of the solution is added from partial oxidation of the 6-phosphogluto a fluorometer tube containing 1 ml of a conate that had been formed from G6P or reagent composed of 50 mM Tris-HCl, pH glucose in previous steps. Fortunately, it was 8.1 (acid:base, l:l), 300 CIM ATP, 1 mM possible to destroy 6-phosphogluconate deMgC&, and 100 j.tM NADP+. hydrogenase in G6PDH from baker’s yeast Step 1 (G6P). After taking an initial readby brief incubation at pH 4.6. The second problem was the slow oxidation of glucose by ing, 0.2 cLg(0.03 U) of G6PDH from baker’s all three commercial types of G6PDH. This yeast is added in a volume < 10 ~1, and a may be a side reaction of the G6PDH en- second reading is made 20 min later. Step 2 (glucose). Without delay, 10 pg of zymes themselves. This caused an error only yeast hexokinase is added in a similar volwhen measuring DG6P, since DG was ume and a third reading is made 20 min always measured together with glucose or later. after glucose had already been converted to Step 3 (NADPH destruction). Again with6-phosphogluconate. The cure was to deout delay, 25 ~1 of 2 M HCl is added and well stroy the glucose with glucose oxidase before mixed. This is followed after at least 10 min the DG6P reaction. Because the oxidase only attacks a-glucose, aldose mutarotase had to by 25 ~1 of 2 M NaOH. Step 4 (2DG + DG6P). After an initial be included also. reading is made, 75 pugof G6PDH are added in a volume of 10 J or less, and a final reading is made 50 min later. MATERIALS AND METHODS Part B. A similar aliquot of the solution is added to 1 ml of reagent composed of the Enzymes, as well as 2-deoxyglucose and same buffer used in Part A (no ATP or Mg), 2-deoxyglucose 6-phosphate, were from plus 100 pM NADP+, 50 pg/ml of glucose Sigma Chemical Co. or Boehringer-Mannoxidase from Aspergilfus niger (about 5 IU), heim. The G6PDH was treated to destroy and 1 pg/ml of aldose mutarotase. contaminating 6-phosphogluconate dehyStep 1 (G6P). The same as step 1 of Part A. drogenase (6PGDH) by incubating for 60 Step 2 (DG6P). Without delay, after the min at 38°C in 50 mM pH 4.6 sodium ace- second reading for Step 1,50 pg of G6PDH is tate buffer (acid:base, 1:l). This was then added and a third reading is made 50 min neutralized with a 0.03 vol of 1 M imidazole later. (The lower level of G6PDH than in base. 6PGDH destruction was partially Step 4 of Part A is sufficient, because of the blocked by levels of (NH&SO4 as low as 0.1 absence of ATP which is somewhat inhibiM. Therefore G6PDH suspensions in strong tory.1 (NH&SO4 were centrifuged and dissolved in Calculations are based on standards and the original volume of the sodium acetate blanks carried through the procedures from buffer for treatment. At least 95% of the the beginning. 2DG is calculated by the dif6PGDH should be removed without loss of ference between the sum of 2DG + DG6P G6PDH. (Part A, Step 4) and DG6P (Part B, Step 2).
510
CHI
ET AL.
Indirect Assay (25 to 500 pmol in Each Aliquot) It is assumed that each aliquot consists of 50 ~1 of a 0.02 M HCl tissue extract.) Part A. Step 1 (G6P). The aliquot is added to a 7 X 75-mm glass tube. To each aliquot is added 50 ~1 of 80 mM Tris-HCl (acid:base, 20:60) containing 600 FM ATP, 2 mM MgC12, 200 pM NADP+, and 0.4 pg/ml of G6PDH and 0.04% bovine serum albumin (BSA). After 20 min a lo-p1 aliquot is transferred into another tube of the same size, which already contains 50 ~1 of 0.025 M NaOH. Step 2 (G6Pplus glucose). Within 10 or 15 min, 1 ~1 of a 1 mg/ml solution of hexokinase is added to the remaining 90 ~1 from Step 1, and after 20 min another IO+1 aliquot is transferred into a tube containing NaOH as in Step 1. Step 3. The NADPH is now destroyed as in the direct assay by first adding 2 ~1 of 2 M HCl to the remaining 80 ~1, followed after at least 10 min by 2 ~1 of 2 M NaOH. Step 4 (2DG + DG6P). To each tube is added 5 ~1 of 1.5 mg/ml G6PDH, and after 50 min a lo-p1 aliquot is transferred into a tube with NaOH as in Step 1. Part B. Step I (G6Pplus DG6P). The 50-~1 aliquot is added to 50 ~1 of 80 mM Tris buffer (acid:base, 20:60) which contains 50 Mg/ml glucose oxidase, 1 p/ml mutarotase, 200 PM NADP+, and 0.02% BSA. After 50 min, 5 ~1 is added of 1 mg/ml G6PDH. After incubation for 40 min, a lo-p1 aliquot is added to a tube with NaOH, as in Part A. Amplification
Procedure
The four tubes with NaOH plus aliquots from Steps 1, 2, and 4 of Part A, and Step 1 of Part B are heated for 5 min in a water bath at about 95°C. A lo-ccl aliquot is added to 100 ~1 of NADP cycling reagent with enzyme levels to give about 2000-fold amplification in 1 h at 25°C. The cycling is terminated by heating for 3 min in a water bath at about 95°C. See Refs. (3) and (4) for details of the
cycling procedure, and the final indicator step to measure the 6-phosphogluconate produced in the cycle. Calculations are based on standards and blanks carried through all steps. Note that although the 1O-p1 aliquots from the different steps represent slightly different fractions of the original sample (because of addition of G6PDH, acid, and alkali), these differences affect the standards and samples alike. To keep the procedures which require amplification as simple as possible, three of the components are calculated indirectly: glucose by subtracting G6P from G6P + glucose, DG6P by subtracting G6P from G6P + DG6P, and DG by subtracting DG6P from DG6P + DG. This is likely to be quite satisfactory for glucose and DG6P, because G6P is usually very low in brain. The calculation of DG by difference may be more subject to error, since in some cases DG6P may equal or exceed DG. Each of these three components can be measured separately if preferred: Glucose and DG6P can be measured directly by first destroying the NADPH from G6P by merely reversing Steps 2 and 3 of parts A and B, respectively. DG direct measurement is more complicated: Step 1. The reagent is the same as for Part A, but G6PDH is increased to give a concentration of 75 pg/ml, and incubation is for 50 min. Step 2. HCl is added as in Step 3 of Part A, but the samples are heated for 5 min at 95°C before neutralizing with NaOH. This is to destroy the high level of G6PDH, as well as the NADPH from G6P and DG6P. Step 3. Hexokinase and G6PDH are added to give concentrations of 10 and 0.2 &ml respectively, with incubation for 20 min. Step 4. The NADPH from glucose is destroyed with HCl, but without heat. This is then neutralized with NaOH as before. Step 5. A high level of GGPDH is added, as in Step 1, to yield NADPH from DG alone.
ENZYMATIC
511
ASSAYS FOR 2-DG AND DG6P
Other Variations in the Assay Aside from greater sensitivity with fluorometric rather than spectrophotometric measurement, the direct assay is simpler in fluorometer tubes, since the long duration of some of the steps would make it awkward to carry out a large experiment in the spectrophotometer without a prohibitive number of cells. However, standardizations are best made spectrophotometrically, and since each component is measured separately there is no need for glucose oxidase and mutarotase. The methods have been adapted and used satisfactorily to measure much smaller quantities than those measured by the procedures given here. Adaptation to measure levels in the 0. I pmol range simply requires reduction in volumes to the microliter and submicroliter level. For these small volumes we have used the oil-well technique (2). Full description of these modifications would be out of place here, but the assay principles and style are the same as presented. EXPERIMENTAL
Kinetics of G6PDH from Baker’s Yeast For analytical applications the levels G6P and DG6P will be well below their spective K,‘s. Therefore, the significant netic value is the first-order rate constant the chosen NADP+ concentration. Table
of rekiat I
gives the first-order rate constants for enzyme levels of 1 &ml, with the NADP+ concentration extrapolated to infinity (&&J. This constant is 1500 times greater for G6P than for DG6P. The difference is due to a combination of a much greater V,,, and a much smaller K,,, (Table I). To achieve 99% conversion kt = 4.6 (i.e., -In O.Ol), where k = kSP X pg/ml G6PDH. Therefore for 99% conversion in 20 min with G6P and in 50 min with DG6P would require a minimum of 4.6/(20 X 2.7) = 0.09 pg/ml G6PDH and 4.6/(50 X 0.00 18) = 5 1 pg/ml G6PDH, respectively. With 0.2 pg/ml G6PDH for 20 min, as recommended for G6P, there is a calculated 0.7% oxidation of DG6P. This is analytically insignificant with levels of the components likely to be encountered in biological material. It is better to err slightly on this side rather than on the other, for the following reason. Glucose will usually be much higher than 2DG. If even a few percent of the G6P from glucose fails to be oxidized at the low G6PDH step, it will cause a large percentage error in the 2DG evaluation. The Kinetics of Hexokinase
with 2DG
The kinetics of baker’s yeast hexokinase were measured with both glucose and ~DG
TABLE 1 KINETICS OF G6PDH FROM BAKER’S YEASTY Constant substrate (PM)
Variable substrate
G6P, 500 G6P, 5 NADP+, 500
NADP+ NADP+ G6P
DG6P, 5 NADP+, 500
NADP + DG6P
Kttl (fiM) 4.4 10.8 19 1.4 540
VILX (firno mg-’ min-‘)
kSp mm (min-‘)
70 2.1 57 0.0018 0.63
n Measurements were made at about 23°C in 50 mM Tris-HCl, pH 8.1, with 0.02% BSA. The G6PDH was about 30% pure. The Km’s are for the variable substrate. V’,,,= is the calculated value for the variable substrate extrapolated to infinity. The constant kg= is the first-order rate constant for G6P or DG6P with 1 &ml of G6PDH and the NADP+ concentration extrapolated to infinity.
512
CHI ET AL. TABLE 2
either glucose or DG in 2 or 3 min with the recommended hexokinase level.
KINETICSOFHEXOKINASE'
Vmm (firno mg-’ min-‘)
Substrate
G’ax (min-‘)
Reproducibility
and Illustrative
Data
In the 1 to 10 nmol, direct-reading range, reproducibility for DG and DG6P is limited only by care in pipetting, instrumental stabil“Measured in 50 mM Tris-HCI buffer, pH 8.1, con- ity, etc, and the coefficient of variation (CV) taining 300 pM ATP at 23’C. The velocities with glucose should not exceed 2%. CV’s in the 10 pmol were followed directly in the fluorometer by the range have averaged 3 to 4%, and in the 0.5 NADPH formed with G6PDH. The 2DG velocities were measured indirectly by stopping the reaction with HCl pmol range have averaged about 6%. In an and measuring the DG6P in a separate step. Because the experiment with 100 ng samples (dry weight) ATP concentration was only about twice the K,,,, the from 4 areas of the hippocampus, 2DG levels true Vmar‘s would be about 50% greater than the V,lmax’~ for quadruplicate samples had an average shown. Vi=, is V,,,,,,/K,,,,and represents the first-order cv of 4.7%. rate constant with a hexokinase concentration of 1 An illustration of the applicability of the a/ml. procedures for all four substances is given in Table 3. Mice were given a convulsive dose under analytical conditions. The I’,,,,, for of pentylenetetrazole (PTZ) 1 min after an Ix; was actually higher than for glucose by a injection of 2DG, and were frozen 0.5 to 3 factor of 2.7 (Table 2). However, the K,,, was min later in liquid N2. Samples of the cerealso higher for DG by an even greater margin bral cortex weighing about 50 mg were dis(5 .Cfold). Therefore, the analytically signifisected out at -20°C and extracted at this cant first-order rate constant is about half as temperature with 1 ml of 0.1 M HCl in absolute methanol. After dilution with aqueous large for 2DG as for glucose. There is clearly no problem in complete phosphorylation of 0.05 M HCl, the samples were centrifuged, Glucose 2DG
111 595
77 210
0.69 0.35
TABLE 3
Min after
2DG
DG
PTZ
1 1.5 1.5
0 0
2 2 3 3 4 4
0.5 0 1 0 2 0 3
Glucose
DG6P
G6P
(rmollkd 448 + 36 445 +40 470+21 454+ 16 379+ 18 459k 9 214+31 437+ 7 248+ 10
180 + 20 251 f 227e 345* 389k 430+31 750+67 477k37 845 f
16 16
I1 36
62
145Ok 134Ok 1540+ 123Ok 115Ok 1610f 79Ok 172Ok 96Ok
170 90 80 40 60 140 40
100 70
115? 16 87+ look 80+ 76? 84+ 65+ 79k 70+
7 9 6 2
11 5 4 8
’ Mice were injected with 1 mmol/kg of 2DG in a tail vein at zero time and with 100 mg/kg of pentylenetetrazole intraperitoneally 1 min later. They were killed by immersion in liquid N2 at the times shown. Each entry is the average & SE for four mice at 1 to 3 min and three mice at 4 min atier 2DG injection.
ENZYMATIC
ASSAYS FOR 2-DG AND DC6P
513
One warning may be in order. In many and the supematants were heated for 5 min at 95°C and analyzed by the direct fluoro- tissues, including the brain, brief ischemia will cause a rapid increase in glycolysis and metric procedure. After a lag period of about 1 min following PTZ injection, there was a therefore conversion of 2DG to DG6P at a dramatic rise in DG6P and fall in 2DG and rate far in excess of that corresponding to glucose, indicating that glucose consumption normal glucose consumption. This possibilhad exceeded the rate of transport of 2DG ity should therefore be kept in mind in applying the proposed enzymatic methods in enand glucose into the brain. ergy metabolism studies. The methods, with appropriate adjustment of the procedures, have been successfully applied to the analysis of brain samples REFERENCES ranging from 50 mg wet wt to 0.1 hg dry wt 1. Sokoloff, L., Reivich, M., Kennedy, C., Des Rosiers, (equal to 0.5 pg wet wt). M. H., Patlak, C. S., Pettigrew, K. D., Sakurada. DISCUSSION
The presentation of the proposed methods has been aimed at measurements in brain in general, and mouse brain in particular. There is, of course, no reason why they are limited to brain. An early 2DG study by Kipnis and Cori was made with rat diaphragm (6), and the Sokoloff procedure has been used with many other types of tissue.
2. 3. 4. 5. 6.
O., and Shinohara, M. (1977) J. Neurochem. 28, 897-916. Lowry, 0. H., and Passonneau, J. V. (1972) A Flexible System of Enzymatic Analysis, Academic Press, New York. Lowry, 0. H. (1980) Mol. Cell. Biochem. 32, 135-146. Chi, M. M.-Y., Lowry, C. V., and Lowry, 0. H. (1978) Anal. Biochem. 128, 186-190. Hintz, C. S., Chi, M. M.-Y., and Lowry, 0. H. (1978) Anal. Biochem. 89, 119-129. Kipnis, D. M., and Cori. C. F. ( 1959) J. Biol. Chem. 234, 171-177.
BIOCHEMISTRY
161,508-5 13 (I 987)
Enzymatic Assays for 2-Deoxyglucose
and 2-Deoxyglucose
6-Phosphate*,’
MAGGIE M.-Y. CHI, MARY ELLEN FLJSATERI,JOYCE G. CARTER, BEVERLY J. NORRIS, DAVID B. MCDOUGAL, JR., AND OLIVER H. LOWRY Departments
of Pharmacology School
and of Neurology and Neurosurgery, Washington of Medicine, St. Louis, Missouri 63110
University
Received November 3. I986 Methods for 2-deoxyglucose (2-M;) and 2-deoxyghtcose 6-phosphate (DG6P) are described which are based on the fact that DG6P is oxidized by glucose-6-phosphate dehydrogenase (G6PDH). but at a rate lOOO-fold slower than for ghrcose 6-phosphate, whereas hexokinase phosphorylates 2DG and glucose at comparable rates. Therefore, by adding the two enzymes in a suitable order, and in appropriate concentrations, 2DG. glucose, DG6P, and glucose 6-P can all be separately measured. To avoid a side reaction from the use of a high level of G6PDH, when measuring DG6P, glucose is first removed with glucose oxidase plus aldose reductase. 0 1987 Academic
Press, Inc.
2-deoxyglucose; 2-deoxyglucose 6-phosphate; glucose-6-phosphate dehydrogenase kinetics; enzymatic assay. KEY
WORDS:
2-Deoxyglucose (2DG)’ has become a very useful tool in the study of brain metabolism. Glucose and 2DG are similarly transported into brain and phosphorylated by hexokinase. However, 2-deoxyglucose 6-phosphate (DG6P) cannot be either converted to an analog of fructose 6-phosphate, or rapidly metabolized otherwise. Therefore, after 2DG administration, nearly all the DG6P that is formed accumulates, and this accumulation can provide a measure of glucose consumption. Because glucose is almost the exclusive fuel for brain, glucose consumption is a useful measure of brain energy metabolism and therefore of neuronal activity. In 1977 Sokoloff et al. described how to exploit this fact, with [14C]2DG, in a classic paper (1). This * This paper is dedicated to the memory of Nathan 0. Kaplan. ’ Supported by grants from the American Cancer Society (BC-4-29). and the U.S. PHS (NS-08862. NS-18387, NS-20762). * Abbreviations used: 2DG, 2-deoxyghrcose; DG6P. 2-deoxyglucose 6-phosphate; G6P, glucose 6-phosphate; G6PDH, glucose-6-phosphate dehydrogenase; 6PGDH, 6-phosphogluconate dehydrogenase; BSA. bovine serum albumin: PTZ, pentylenetetrazole. 0003-2697187 $3.00 Copyright Q 1987 by Academic Press, Inc. Al! rights of reproduction in any form reserved.
508
now widely used radioautographic method requires a 30- to 45-min time lapse after 2DG injection for dissipation of most of the unphosphorylated 2DG. The time lag limits the method to the study of events which occur on a long time scale. This report describes sensitive enzymatic methods for measuring both 2DG and 2DG6P as well as glucose and glucose 6phosphate (G6P) in the same tissue sample. With these methods, it has proven possible to measure changes in brain glucose consumption on a time scale of 1 to 5 min. The methods are based on the fact that both G6P and DG6P can be oxidized by G6P dehydrogenase (G6PDH) but at rates that differ by a factor of more than a thousand. Therefore, the NADH or NADPH produced with a low level of enzyme is a measure of G6P, while that produced with a much higher G6PDH level is DG6P. Glucose and 2DG are similarly distinguished after phosphorylation by hexokinase. For higher levels of these compounds (0.5 nmol or more) the fluorescence of the NADPH generated is measured directly. For lower levels (down to a few femtomoles if
ENZYMATIC
ASSAYS
FOR
2-DG
AND
DG6P
509
necessary), the NADPH is amplified by en- Direct Assays (0.5 to IO nmol in Each Aliquot) zymatic cycling (2,3). Two problems were encountered, both resulting from the use of such a high level of The analysis of a solution such as a tissue G6PDH. Commercial preparations of extract which contains all four components, G6PDH almost invariably contained traces 2DG, 2DG6P, glucose, and G6P, is made in of 6-phosphogluconate dehydrogenase. This two parts. caused errors in DG and DG6P readings Part A. An aliquot of the solution is added from partial oxidation of the 6-phosphogluto a fluorometer tube containing 1 ml of a conate that had been formed from G6P or reagent composed of 50 mM Tris-HCl, pH glucose in previous steps. Fortunately, it was 8.1 (acid:base, l:l), 300 CIM ATP, 1 mM possible to destroy 6-phosphogluconate deMgC&, and 100 j.tM NADP+. hydrogenase in G6PDH from baker’s yeast Step 1 (G6P). After taking an initial readby brief incubation at pH 4.6. The second problem was the slow oxidation of glucose by ing, 0.2 cLg(0.03 U) of G6PDH from baker’s all three commercial types of G6PDH. This yeast is added in a volume < 10 ~1, and a may be a side reaction of the G6PDH en- second reading is made 20 min later. Step 2 (glucose). Without delay, 10 pg of zymes themselves. This caused an error only yeast hexokinase is added in a similar volwhen measuring DG6P, since DG was ume and a third reading is made 20 min always measured together with glucose or later. after glucose had already been converted to Step 3 (NADPH destruction). Again with6-phosphogluconate. The cure was to deout delay, 25 ~1 of 2 M HCl is added and well stroy the glucose with glucose oxidase before mixed. This is followed after at least 10 min the DG6P reaction. Because the oxidase only attacks a-glucose, aldose mutarotase had to by 25 ~1 of 2 M NaOH. Step 4 (2DG + DG6P). After an initial be included also. reading is made, 75 pugof G6PDH are added in a volume of 10 J or less, and a final reading is made 50 min later. MATERIALS AND METHODS Part B. A similar aliquot of the solution is added to 1 ml of reagent composed of the Enzymes, as well as 2-deoxyglucose and same buffer used in Part A (no ATP or Mg), 2-deoxyglucose 6-phosphate, were from plus 100 pM NADP+, 50 pg/ml of glucose Sigma Chemical Co. or Boehringer-Mannoxidase from Aspergilfus niger (about 5 IU), heim. The G6PDH was treated to destroy and 1 pg/ml of aldose mutarotase. contaminating 6-phosphogluconate dehyStep 1 (G6P). The same as step 1 of Part A. drogenase (6PGDH) by incubating for 60 Step 2 (DG6P). Without delay, after the min at 38°C in 50 mM pH 4.6 sodium ace- second reading for Step 1,50 pg of G6PDH is tate buffer (acid:base, 1:l). This was then added and a third reading is made 50 min neutralized with a 0.03 vol of 1 M imidazole later. (The lower level of G6PDH than in base. 6PGDH destruction was partially Step 4 of Part A is sufficient, because of the blocked by levels of (NH&SO4 as low as 0.1 absence of ATP which is somewhat inhibiM. Therefore G6PDH suspensions in strong tory.1 (NH&SO4 were centrifuged and dissolved in Calculations are based on standards and the original volume of the sodium acetate blanks carried through the procedures from buffer for treatment. At least 95% of the the beginning. 2DG is calculated by the dif6PGDH should be removed without loss of ference between the sum of 2DG + DG6P G6PDH. (Part A, Step 4) and DG6P (Part B, Step 2).
510
CHI
ET AL.
Indirect Assay (25 to 500 pmol in Each Aliquot) It is assumed that each aliquot consists of 50 ~1 of a 0.02 M HCl tissue extract.) Part A. Step 1 (G6P). The aliquot is added to a 7 X 75-mm glass tube. To each aliquot is added 50 ~1 of 80 mM Tris-HCl (acid:base, 20:60) containing 600 FM ATP, 2 mM MgC12, 200 pM NADP+, and 0.4 pg/ml of G6PDH and 0.04% bovine serum albumin (BSA). After 20 min a lo-p1 aliquot is transferred into another tube of the same size, which already contains 50 ~1 of 0.025 M NaOH. Step 2 (G6Pplus glucose). Within 10 or 15 min, 1 ~1 of a 1 mg/ml solution of hexokinase is added to the remaining 90 ~1 from Step 1, and after 20 min another IO+1 aliquot is transferred into a tube containing NaOH as in Step 1. Step 3. The NADPH is now destroyed as in the direct assay by first adding 2 ~1 of 2 M HCl to the remaining 80 ~1, followed after at least 10 min by 2 ~1 of 2 M NaOH. Step 4 (2DG + DG6P). To each tube is added 5 ~1 of 1.5 mg/ml G6PDH, and after 50 min a lo-p1 aliquot is transferred into a tube with NaOH as in Step 1. Part B. Step I (G6Pplus DG6P). The 50-~1 aliquot is added to 50 ~1 of 80 mM Tris buffer (acid:base, 20:60) which contains 50 Mg/ml glucose oxidase, 1 p/ml mutarotase, 200 PM NADP+, and 0.02% BSA. After 50 min, 5 ~1 is added of 1 mg/ml G6PDH. After incubation for 40 min, a lo-p1 aliquot is added to a tube with NaOH, as in Part A. Amplification
Procedure
The four tubes with NaOH plus aliquots from Steps 1, 2, and 4 of Part A, and Step 1 of Part B are heated for 5 min in a water bath at about 95°C. A lo-ccl aliquot is added to 100 ~1 of NADP cycling reagent with enzyme levels to give about 2000-fold amplification in 1 h at 25°C. The cycling is terminated by heating for 3 min in a water bath at about 95°C. See Refs. (3) and (4) for details of the
cycling procedure, and the final indicator step to measure the 6-phosphogluconate produced in the cycle. Calculations are based on standards and blanks carried through all steps. Note that although the 1O-p1 aliquots from the different steps represent slightly different fractions of the original sample (because of addition of G6PDH, acid, and alkali), these differences affect the standards and samples alike. To keep the procedures which require amplification as simple as possible, three of the components are calculated indirectly: glucose by subtracting G6P from G6P + glucose, DG6P by subtracting G6P from G6P + DG6P, and DG by subtracting DG6P from DG6P + DG. This is likely to be quite satisfactory for glucose and DG6P, because G6P is usually very low in brain. The calculation of DG by difference may be more subject to error, since in some cases DG6P may equal or exceed DG. Each of these three components can be measured separately if preferred: Glucose and DG6P can be measured directly by first destroying the NADPH from G6P by merely reversing Steps 2 and 3 of parts A and B, respectively. DG direct measurement is more complicated: Step 1. The reagent is the same as for Part A, but G6PDH is increased to give a concentration of 75 pg/ml, and incubation is for 50 min. Step 2. HCl is added as in Step 3 of Part A, but the samples are heated for 5 min at 95°C before neutralizing with NaOH. This is to destroy the high level of G6PDH, as well as the NADPH from G6P and DG6P. Step 3. Hexokinase and G6PDH are added to give concentrations of 10 and 0.2 &ml respectively, with incubation for 20 min. Step 4. The NADPH from glucose is destroyed with HCl, but without heat. This is then neutralized with NaOH as before. Step 5. A high level of GGPDH is added, as in Step 1, to yield NADPH from DG alone.
ENZYMATIC
511
ASSAYS FOR 2-DG AND DG6P
Other Variations in the Assay Aside from greater sensitivity with fluorometric rather than spectrophotometric measurement, the direct assay is simpler in fluorometer tubes, since the long duration of some of the steps would make it awkward to carry out a large experiment in the spectrophotometer without a prohibitive number of cells. However, standardizations are best made spectrophotometrically, and since each component is measured separately there is no need for glucose oxidase and mutarotase. The methods have been adapted and used satisfactorily to measure much smaller quantities than those measured by the procedures given here. Adaptation to measure levels in the 0. I pmol range simply requires reduction in volumes to the microliter and submicroliter level. For these small volumes we have used the oil-well technique (2). Full description of these modifications would be out of place here, but the assay principles and style are the same as presented. EXPERIMENTAL
Kinetics of G6PDH from Baker’s Yeast For analytical applications the levels G6P and DG6P will be well below their spective K,‘s. Therefore, the significant netic value is the first-order rate constant the chosen NADP+ concentration. Table
of rekiat I
gives the first-order rate constants for enzyme levels of 1 &ml, with the NADP+ concentration extrapolated to infinity (&&J. This constant is 1500 times greater for G6P than for DG6P. The difference is due to a combination of a much greater V,,, and a much smaller K,,, (Table I). To achieve 99% conversion kt = 4.6 (i.e., -In O.Ol), where k = kSP X pg/ml G6PDH. Therefore for 99% conversion in 20 min with G6P and in 50 min with DG6P would require a minimum of 4.6/(20 X 2.7) = 0.09 pg/ml G6PDH and 4.6/(50 X 0.00 18) = 5 1 pg/ml G6PDH, respectively. With 0.2 pg/ml G6PDH for 20 min, as recommended for G6P, there is a calculated 0.7% oxidation of DG6P. This is analytically insignificant with levels of the components likely to be encountered in biological material. It is better to err slightly on this side rather than on the other, for the following reason. Glucose will usually be much higher than 2DG. If even a few percent of the G6P from glucose fails to be oxidized at the low G6PDH step, it will cause a large percentage error in the 2DG evaluation. The Kinetics of Hexokinase
with 2DG
The kinetics of baker’s yeast hexokinase were measured with both glucose and ~DG
TABLE 1 KINETICS OF G6PDH FROM BAKER’S YEASTY Constant substrate (PM)
Variable substrate
G6P, 500 G6P, 5 NADP+, 500
NADP+ NADP+ G6P
DG6P, 5 NADP+, 500
NADP + DG6P
Kttl (fiM) 4.4 10.8 19 1.4 540
VILX (firno mg-’ min-‘)
kSp mm (min-‘)
70 2.1 57 0.0018 0.63
n Measurements were made at about 23°C in 50 mM Tris-HCl, pH 8.1, with 0.02% BSA. The G6PDH was about 30% pure. The Km’s are for the variable substrate. V’,,,= is the calculated value for the variable substrate extrapolated to infinity. The constant kg= is the first-order rate constant for G6P or DG6P with 1 &ml of G6PDH and the NADP+ concentration extrapolated to infinity.
512
CHI ET AL. TABLE 2
either glucose or DG in 2 or 3 min with the recommended hexokinase level.
KINETICSOFHEXOKINASE'
Vmm (firno mg-’ min-‘)
Substrate
G’ax (min-‘)
Reproducibility
and Illustrative
Data
In the 1 to 10 nmol, direct-reading range, reproducibility for DG and DG6P is limited only by care in pipetting, instrumental stabil“Measured in 50 mM Tris-HCI buffer, pH 8.1, con- ity, etc, and the coefficient of variation (CV) taining 300 pM ATP at 23’C. The velocities with glucose should not exceed 2%. CV’s in the 10 pmol were followed directly in the fluorometer by the range have averaged 3 to 4%, and in the 0.5 NADPH formed with G6PDH. The 2DG velocities were measured indirectly by stopping the reaction with HCl pmol range have averaged about 6%. In an and measuring the DG6P in a separate step. Because the experiment with 100 ng samples (dry weight) ATP concentration was only about twice the K,,,, the from 4 areas of the hippocampus, 2DG levels true Vmar‘s would be about 50% greater than the V,lmax’~ for quadruplicate samples had an average shown. Vi=, is V,,,,,,/K,,,,and represents the first-order cv of 4.7%. rate constant with a hexokinase concentration of 1 An illustration of the applicability of the a/ml. procedures for all four substances is given in Table 3. Mice were given a convulsive dose under analytical conditions. The I’,,,,, for of pentylenetetrazole (PTZ) 1 min after an Ix; was actually higher than for glucose by a injection of 2DG, and were frozen 0.5 to 3 factor of 2.7 (Table 2). However, the K,,, was min later in liquid N2. Samples of the cerealso higher for DG by an even greater margin bral cortex weighing about 50 mg were dis(5 .Cfold). Therefore, the analytically signifisected out at -20°C and extracted at this cant first-order rate constant is about half as temperature with 1 ml of 0.1 M HCl in absolute methanol. After dilution with aqueous large for 2DG as for glucose. There is clearly no problem in complete phosphorylation of 0.05 M HCl, the samples were centrifuged, Glucose 2DG
111 595
77 210
0.69 0.35
TABLE 3
Min after
2DG
DG
PTZ
1 1.5 1.5
0 0
2 2 3 3 4 4
0.5 0 1 0 2 0 3
Glucose
DG6P
G6P
(rmollkd 448 + 36 445 +40 470+21 454+ 16 379+ 18 459k 9 214+31 437+ 7 248+ 10
180 + 20 251 f 227e 345* 389k 430+31 750+67 477k37 845 f
16 16
I1 36
62
145Ok 134Ok 1540+ 123Ok 115Ok 1610f 79Ok 172Ok 96Ok
170 90 80 40 60 140 40
100 70
115? 16 87+ look 80+ 76? 84+ 65+ 79k 70+
7 9 6 2
11 5 4 8
’ Mice were injected with 1 mmol/kg of 2DG in a tail vein at zero time and with 100 mg/kg of pentylenetetrazole intraperitoneally 1 min later. They were killed by immersion in liquid N2 at the times shown. Each entry is the average & SE for four mice at 1 to 3 min and three mice at 4 min atier 2DG injection.
ENZYMATIC
ASSAYS FOR 2-DG AND DC6P
513
One warning may be in order. In many and the supematants were heated for 5 min at 95°C and analyzed by the direct fluoro- tissues, including the brain, brief ischemia will cause a rapid increase in glycolysis and metric procedure. After a lag period of about 1 min following PTZ injection, there was a therefore conversion of 2DG to DG6P at a dramatic rise in DG6P and fall in 2DG and rate far in excess of that corresponding to glucose, indicating that glucose consumption normal glucose consumption. This possibilhad exceeded the rate of transport of 2DG ity should therefore be kept in mind in applying the proposed enzymatic methods in enand glucose into the brain. ergy metabolism studies. The methods, with appropriate adjustment of the procedures, have been successfully applied to the analysis of brain samples REFERENCES ranging from 50 mg wet wt to 0.1 hg dry wt 1. Sokoloff, L., Reivich, M., Kennedy, C., Des Rosiers, (equal to 0.5 pg wet wt). M. H., Patlak, C. S., Pettigrew, K. D., Sakurada. DISCUSSION
The presentation of the proposed methods has been aimed at measurements in brain in general, and mouse brain in particular. There is, of course, no reason why they are limited to brain. An early 2DG study by Kipnis and Cori was made with rat diaphragm (6), and the Sokoloff procedure has been used with many other types of tissue.
2. 3. 4. 5. 6.
O., and Shinohara, M. (1977) J. Neurochem. 28, 897-916. Lowry, 0. H., and Passonneau, J. V. (1972) A Flexible System of Enzymatic Analysis, Academic Press, New York. Lowry, 0. H. (1980) Mol. Cell. Biochem. 32, 135-146. Chi, M. M.-Y., Lowry, C. V., and Lowry, 0. H. (1978) Anal. Biochem. 128, 186-190. Hintz, C. S., Chi, M. M.-Y., and Lowry, 0. H. (1978) Anal. Biochem. 89, 119-129. Kipnis, D. M., and Cori. C. F. ( 1959) J. Biol. Chem. 234, 171-177.