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Enzymatic lactate-specific radioactivity determination in biological samples
ANALYTICAL
BIOCHEMISTRY
148,
190-193 (1985)
Enzymatic Lactate-Specific Radioactivity in Biological Samples’ P. RDCA, M. GIANOTTI, Departament
de Bioquimica, 07071
Determination
AND A. PALOU
Facultat de Ci&cies, Universitat de Palma Palma de Mallorca, Balears, Spain
de Mallorca,
Received January 22, 1985 A method for the measurement of specific lactate radioactivity in biological samples is presented. It is based on the following steps: (a) enzymatic conversion of lactate to pyruvate, (b) pyruvate conversion to 2,4dinitrophenylhydrazone, (c) concentration-separation of the latter in reusable Amberlite XAD-7 polymeric adsorbent columns, and finally (d) estimation of the radioactivity thus retained compared with that of enzymatically untreated aliquots of the same samples. Specificity was ensured by the use of lactate dehydrogenase as specific recognizing agent for lactic acid. No interference from glucose, lactate, or amino acids was observed. The method presented is simple and can be applied in routine multiple estimations of lactic acid radioactivity in coniunction with the enzymatic measurement of lactate in biological samples in tracer metabolic studies. Q 1985 Academic Press, Inc.
Several methods are currently used for lactate specific radioactivity determination in biological samples. These are based on ionexchange (l), thin layer (2), or partition (3) chromatography separation from other materials in previously deproteinized samples. Oxidation-degradation-based methods are also used (3-5) despite possible free-pyruvate interference (4). The selective chemical formation of lactate derivatives (3) followed by solvent extraction and purification previous to radioactivity measurement has also been postulated. Other specific methods are based on the oxidation of L-lactate with lactate dehydrogenase followed by pyruvate derivatization, purification, and radioactivity estimation (6,7). However, high oscillations in the mean recovery and poor repetitiveness have been presented as main drawbacks of these methods (8). Methods based on the use of the reverse-isotope-dilution principle (8-
10) usually yield accurate results, but are too time consuming and thus inadequate for manifold experiments. The use of polymeric porous hydrophobic adsorbing beads, such as Amberlites XAD, for trapping trace amounts of organic compounds has improved the solvent extraction procedures ( 1 l- 13). We had already applied Amberlites to the estimation of 14C label in pyruvate (12) and amino acids (13). In this paper we present a method for lactate-specific radioactivity determination based on the selective (enzymatic) transformation of lactate to pyruvate, coupled to the formation of the 2,4dinitrophenyl hydrazone derivative, and quantitative trapping in a single purification step in Amberlite XAD-7 columns followed by radioactivity counting of the lipophilic eluate. MATERIALS
Copyright Q 1985 by Academic Press. Inc. All rights of reproduction in any form reserved.
METHODS
All reagents used were of analytical grade. Labeled materials were obtained from The Radiochemical Center (Amersham, U. IL). Biochemical standards and reagents were ob-
’ Supported by a grant from the “Comision Asesora para la Investigation Cientifica y Tkxica” from the Government of Spain and a grant from the University of Palma de Mallorca. 0003-2697185 $3.00
AND
190
ENZYMATIC
LACTATE-SPECIFIC
tamed from Sigma Chemical Company (St. Louis, MO.). Samples of rat blood, plasma or standard solutions containing known amounts of L[U-‘4C]lactic acid [sodium salt (from 0.22 to 11.1 mM; final specific radioactivity of 15.1 to 3.1 MBq/mmol)] were used. Labeled lactate was also added to plasma or solutions containing the following U-14C-labeled materials used to validate the method: 0.21 mM glycerol (36.7 MBq/mmol), 0.2 mM pyruvate (34.0 MBq/mmol), 5 mM glucose (8.32 MBq/ mmol), 1.01 mM L-alanine (7.28 MBq/ mmol), 1.00 mM L-glutamate (7.36 MBq/ mmol), 1.02 mM glycine (7.21 MBq/mmol), and 0.99 mM L-serine (7.40 MBq/mmol) both individually and in mixed solutions. The metacrylate-based hydrophobic adsorbing resin Amberlite XAD-7 (Sigma) was used because its hydrophobicity was lower in the Amberlite XAD series and it is able to retain even mildly hydrophylic materials with hydrophobic domains in the molecule. Because materials are attached less effectively, the elution procedure is easier than with other strong resins. One milliliter of sample was deproteinized with 1 ml 12% (w/v) perchloric acid. Supernatants were then neutralized with 0.35 ml of 2 N KOH containing 2 N KHC03 and centrifuged again. Two aliquots of 0.3 ml from each final supematant were used. The first (A) was incubated with 1.20 ml of 537 mM glycine/hydrazine hydrate buffer (pH 9.0) containing 2.75 mM NAD+ and excess lactate dehydrogenase (14). The NADH 334nm extinction increase was also measured and used to calculate the lactate content of the sample. The second aliquot (B) was incubated under the same conditions but with no enzyme. Then both aliquots were processed in parallel: 1.4 ml of each was treated for 20 min at 25°C with 0.5 ml of 1 g/liter 2,4-dinitrophenylhydrazine in 2 N HCl. One milliliter of each of these samples was then passed through small plastic columns containing 1 g of Amberlite XAD-7 (13) (previously washed with acetone and equilibrated
RADIOACTIVITY
191
DETERMINATION
with up to 6 ml of 15% ethanol to eliminate acetone; the columns can be recycled several times after acetone washing). The samples were retained for about 2-3 min before discharging and washing with 15% ethanol (fractions discarded). Finally 6 ml of acetone was used to elute the pyruvate-2,4-dinitrophenylhydrazone that had formed. These extracts were dried and then dissolved in 5 ml of 2,5-diphenyloxazole- 1,4-bis(5-phenyloxazol-2-yl)benzene-xylene liquid scintillation cocktail for radioactivity estimation. The difference between the radioactivities found in both parallel sample aliquots (A and B) gave the lactate radioactivity. The lactate-specific radioactivity could then be calculated. RESULTS
AND
DISCUSSION
Table 1 shows the relation between lactate radioactivity present in the samples (standards, blood, or plasma) versus radioactivity found in the acetone eluate with the method described. The lineal correlation coefficient of added versus found radioactivity was 0.999 (n = 10). The mean percentage recovery of lactate radioactivity with the method described was 83.5 -t 0.5 (n = 10). No differences were observed between the recoveries of [r4C]lactate standard solutions (83.1 +- 1.3, 12 = 4) and those of rat plasma (83.3 + 0.9, n = 2) or blood (84.0 f 0.6, n = 4) samples tainted with known amounts of [‘4C]lactate, irrespective of the presence of other labeled materials. The formation of pyruvate-2,4dinitrophenylhydrazone under the conditions tested was not complete (12); thus there was a mean 16.3% loss of radioactivity in the whole derivatization/purification process that must be taken into account in the calculation of the original lactate radioactivity. The high constancy of this figure under the conditions described made superfIuous the inclusion of known standard solutions to correct each batch of samples studied at the same time. The repetitiveness of the method was studied by measuring the lactate specific radioactivity in a set of three different frozen samples, defrosted and assayed on different days. The
192
RGCA,
GIANOTTI,
AND
TABLE DISTRIBUTION
PALOU
1
AND RECOVERIES OF 14C-L~~~~~~ RADIOACTIVITY AND OTHER *%-LABELED DIFFERENT FRACX’IONS OBTAINED WITH THE METHOD DESCRIBED Radioactivity
Added (4
Standard solution Lactate ( 12.4) Lactate (24.9) Lactate (49.8) Lactate (62.2) Mean
recovery
Blood Lactate Lactate Lactate
(27.1) (44.3) (74.9)
Mean
recovery
Plasma Lactate Lactate
(32.9) (83.1)
Mean
recovery
193 193 193 193
Recovered in acetone eluate
Individual
26.9 36.3 32.4 30.0
Untreated
LDHtreated (A)
Untreated (B)
190.3 190.4 189.4 190.3
166.9 158.1 161.5 168.4
3.09 2.51 3.38 2.80
Difference (A - B)
163.8 156.1 158.2 165.6
Final recovery of added label as Y-lactate (A - B) x 100/a
84.5 80.1 81.9 85.8 83.2 + 1.2
193 193 193
32.1 34.0 31.3
189.3 190.8 189.9
189.3 165.6 167.0
2.90 2.51 2.20
163.1 164.8
82.9 84.5 85.4 84.6 f 0.6
193 193
31.3 31.4
189.7 189.9
162.5 165.6
3.08 3.67
159.3 161.9
82.6 83.9 83.3 + 0.9
Standard solutions Pyruvate (11.3) Glucose (282) Glycerol ( 11.8) L-Alanine (56.9) L-Glutamate (56.3) Glycine (57.5) L-Set-me (56.0) Note.
LDHtreated
AMONG
(Bq)
Recovered in ethanol fraction U-W Samples (put on column, nmol)
MATERIALS
values
382 2348 426 414 415 414 414 shown
51.6 2305 419 410 405 410 406 were the means
50.6 2312 418 410 407 410 406
329.9 32.3 7.7 5.1 8.8 4.3 6.4
of duplicate
determinations.
mean standard deviation of the measurements (n = 5 set) was 1.3% of the mean values measured, thus indicating a high degree of repeatability in the process described, in the same range of repeatability as the actual cold lactate spectrophotometric enzymatic measurement ( 1.4%). The actual recovery of other labeled materials is also shown in Table 1. They were tested at concentrations comparable to those found in the rat’s biological fluids and were the most commonly plausible
331.0 30.0 8.0 4.6 8.0 4.4 5.6
1.1 2.3 -0.3 0.5 0.8 -0.1 0.9
0.3 0.1 -0. I 0.1 0.2 -0. I 0.2
contaminants that could interfere in experiments in which labeled lactate is thought to be present (12,13,15,16). No interferences from glucose, pyruvate, or the amino acids tested were observed. Actually, only minor interferences could be expected because the procedure described ensures a high degree of specificity at the enzymatic level and eliminates other interferences through the use of its own blank. This method shows high repetitiveness, is
ENZYMATIC
LACTATE-SPECIFIC
relatively inexpensive, and is applicable to large numbers of samples for the determination of lactate-specific radioactivity in tracer metabolic studies. REFERENCES 1. Katz, J., Okajima, F., Chenoweth, M., and Dunn, A. (1981) Biochem. J. 194, 513-524. 2. Leichtweib, H. P., and Schroder, H. (1981) Pflugers Arch.
390, 80-85.
3. Bernstein, I. A., and Woot, H. G. (1957) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 4, pp. 561-584. Academic Press, New York. 4. Eldridge, F. L., T’so, L., and Chang, H. (1974) J. Appl. Physiol.
37, 3 16-320.
5. Roseman, J. (1953) Amer. Chem. Sot. 75, 3854. 6. Jorfeldt, L. (1970) AC?Q Physiol. Scund. (Suppl) 338, 1.
RADIOACTIVITY
193
DETERMINATION
7. Kusaka, N., and Vi, M. (1973) Anal. Biochem. 52, 369. 8. Relly, P. E. B. (1975) Anal. Biochem. 64, 37-44. 9. Annisol, E. F., Lindsay, D. B., and White, R. R. (1963)
Biochem.
J. 88, 243.
10. Long, C. L., Mashima, J., and Gump, F. (1971) Anal.
Biochem.
40, 386.
11. Dressier, M. (1979) J. Chromatogr. 165, 167-207. 12. Gianotti, M., Rota, P., and Palou, A. (1984) J. Biochem. Biophys. Methods 10, 181-185. 13. Rota, P., Palou, A., and Alemany, M. (1983) J. Biochem.
Biophys.
Methods
8, 63-67.
14. Gutmann, I., and Wahlefeld, A. W. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H. U., ed.), Vol. 3, pp. 1464-1468, Academic Press, New York. 15. Alemany, M., Palou, A., Codina, J., and Herrera, E. (1978) Diabete Metab. 4, 181-186. 16. Okajima, F., Chenoweth, M., Rognstad, R., and Dunn, A. (1981) Biochem. J. 194, 525-540.
BIOCHEMISTRY
148,
190-193 (1985)
Enzymatic Lactate-Specific Radioactivity in Biological Samples’ P. RDCA, M. GIANOTTI, Departament
de Bioquimica, 07071
Determination
AND A. PALOU
Facultat de Ci&cies, Universitat de Palma Palma de Mallorca, Balears, Spain
de Mallorca,
Received January 22, 1985 A method for the measurement of specific lactate radioactivity in biological samples is presented. It is based on the following steps: (a) enzymatic conversion of lactate to pyruvate, (b) pyruvate conversion to 2,4dinitrophenylhydrazone, (c) concentration-separation of the latter in reusable Amberlite XAD-7 polymeric adsorbent columns, and finally (d) estimation of the radioactivity thus retained compared with that of enzymatically untreated aliquots of the same samples. Specificity was ensured by the use of lactate dehydrogenase as specific recognizing agent for lactic acid. No interference from glucose, lactate, or amino acids was observed. The method presented is simple and can be applied in routine multiple estimations of lactic acid radioactivity in coniunction with the enzymatic measurement of lactate in biological samples in tracer metabolic studies. Q 1985 Academic Press, Inc.
Several methods are currently used for lactate specific radioactivity determination in biological samples. These are based on ionexchange (l), thin layer (2), or partition (3) chromatography separation from other materials in previously deproteinized samples. Oxidation-degradation-based methods are also used (3-5) despite possible free-pyruvate interference (4). The selective chemical formation of lactate derivatives (3) followed by solvent extraction and purification previous to radioactivity measurement has also been postulated. Other specific methods are based on the oxidation of L-lactate with lactate dehydrogenase followed by pyruvate derivatization, purification, and radioactivity estimation (6,7). However, high oscillations in the mean recovery and poor repetitiveness have been presented as main drawbacks of these methods (8). Methods based on the use of the reverse-isotope-dilution principle (8-
10) usually yield accurate results, but are too time consuming and thus inadequate for manifold experiments. The use of polymeric porous hydrophobic adsorbing beads, such as Amberlites XAD, for trapping trace amounts of organic compounds has improved the solvent extraction procedures ( 1 l- 13). We had already applied Amberlites to the estimation of 14C label in pyruvate (12) and amino acids (13). In this paper we present a method for lactate-specific radioactivity determination based on the selective (enzymatic) transformation of lactate to pyruvate, coupled to the formation of the 2,4dinitrophenyl hydrazone derivative, and quantitative trapping in a single purification step in Amberlite XAD-7 columns followed by radioactivity counting of the lipophilic eluate. MATERIALS
Copyright Q 1985 by Academic Press. Inc. All rights of reproduction in any form reserved.
METHODS
All reagents used were of analytical grade. Labeled materials were obtained from The Radiochemical Center (Amersham, U. IL). Biochemical standards and reagents were ob-
’ Supported by a grant from the “Comision Asesora para la Investigation Cientifica y Tkxica” from the Government of Spain and a grant from the University of Palma de Mallorca. 0003-2697185 $3.00
AND
190
ENZYMATIC
LACTATE-SPECIFIC
tamed from Sigma Chemical Company (St. Louis, MO.). Samples of rat blood, plasma or standard solutions containing known amounts of L[U-‘4C]lactic acid [sodium salt (from 0.22 to 11.1 mM; final specific radioactivity of 15.1 to 3.1 MBq/mmol)] were used. Labeled lactate was also added to plasma or solutions containing the following U-14C-labeled materials used to validate the method: 0.21 mM glycerol (36.7 MBq/mmol), 0.2 mM pyruvate (34.0 MBq/mmol), 5 mM glucose (8.32 MBq/ mmol), 1.01 mM L-alanine (7.28 MBq/ mmol), 1.00 mM L-glutamate (7.36 MBq/ mmol), 1.02 mM glycine (7.21 MBq/mmol), and 0.99 mM L-serine (7.40 MBq/mmol) both individually and in mixed solutions. The metacrylate-based hydrophobic adsorbing resin Amberlite XAD-7 (Sigma) was used because its hydrophobicity was lower in the Amberlite XAD series and it is able to retain even mildly hydrophylic materials with hydrophobic domains in the molecule. Because materials are attached less effectively, the elution procedure is easier than with other strong resins. One milliliter of sample was deproteinized with 1 ml 12% (w/v) perchloric acid. Supernatants were then neutralized with 0.35 ml of 2 N KOH containing 2 N KHC03 and centrifuged again. Two aliquots of 0.3 ml from each final supematant were used. The first (A) was incubated with 1.20 ml of 537 mM glycine/hydrazine hydrate buffer (pH 9.0) containing 2.75 mM NAD+ and excess lactate dehydrogenase (14). The NADH 334nm extinction increase was also measured and used to calculate the lactate content of the sample. The second aliquot (B) was incubated under the same conditions but with no enzyme. Then both aliquots were processed in parallel: 1.4 ml of each was treated for 20 min at 25°C with 0.5 ml of 1 g/liter 2,4-dinitrophenylhydrazine in 2 N HCl. One milliliter of each of these samples was then passed through small plastic columns containing 1 g of Amberlite XAD-7 (13) (previously washed with acetone and equilibrated
RADIOACTIVITY
191
DETERMINATION
with up to 6 ml of 15% ethanol to eliminate acetone; the columns can be recycled several times after acetone washing). The samples were retained for about 2-3 min before discharging and washing with 15% ethanol (fractions discarded). Finally 6 ml of acetone was used to elute the pyruvate-2,4-dinitrophenylhydrazone that had formed. These extracts were dried and then dissolved in 5 ml of 2,5-diphenyloxazole- 1,4-bis(5-phenyloxazol-2-yl)benzene-xylene liquid scintillation cocktail for radioactivity estimation. The difference between the radioactivities found in both parallel sample aliquots (A and B) gave the lactate radioactivity. The lactate-specific radioactivity could then be calculated. RESULTS
AND
DISCUSSION
Table 1 shows the relation between lactate radioactivity present in the samples (standards, blood, or plasma) versus radioactivity found in the acetone eluate with the method described. The lineal correlation coefficient of added versus found radioactivity was 0.999 (n = 10). The mean percentage recovery of lactate radioactivity with the method described was 83.5 -t 0.5 (n = 10). No differences were observed between the recoveries of [r4C]lactate standard solutions (83.1 +- 1.3, 12 = 4) and those of rat plasma (83.3 + 0.9, n = 2) or blood (84.0 f 0.6, n = 4) samples tainted with known amounts of [‘4C]lactate, irrespective of the presence of other labeled materials. The formation of pyruvate-2,4dinitrophenylhydrazone under the conditions tested was not complete (12); thus there was a mean 16.3% loss of radioactivity in the whole derivatization/purification process that must be taken into account in the calculation of the original lactate radioactivity. The high constancy of this figure under the conditions described made superfIuous the inclusion of known standard solutions to correct each batch of samples studied at the same time. The repetitiveness of the method was studied by measuring the lactate specific radioactivity in a set of three different frozen samples, defrosted and assayed on different days. The
192
RGCA,
GIANOTTI,
AND
TABLE DISTRIBUTION
PALOU
1
AND RECOVERIES OF 14C-L~~~~~~ RADIOACTIVITY AND OTHER *%-LABELED DIFFERENT FRACX’IONS OBTAINED WITH THE METHOD DESCRIBED Radioactivity
Added (4
Standard solution Lactate ( 12.4) Lactate (24.9) Lactate (49.8) Lactate (62.2) Mean
recovery
Blood Lactate Lactate Lactate
(27.1) (44.3) (74.9)
Mean
recovery
Plasma Lactate Lactate
(32.9) (83.1)
Mean
recovery
193 193 193 193
Recovered in acetone eluate
Individual
26.9 36.3 32.4 30.0
Untreated
LDHtreated (A)
Untreated (B)
190.3 190.4 189.4 190.3
166.9 158.1 161.5 168.4
3.09 2.51 3.38 2.80
Difference (A - B)
163.8 156.1 158.2 165.6
Final recovery of added label as Y-lactate (A - B) x 100/a
84.5 80.1 81.9 85.8 83.2 + 1.2
193 193 193
32.1 34.0 31.3
189.3 190.8 189.9
189.3 165.6 167.0
2.90 2.51 2.20
163.1 164.8
82.9 84.5 85.4 84.6 f 0.6
193 193
31.3 31.4
189.7 189.9
162.5 165.6
3.08 3.67
159.3 161.9
82.6 83.9 83.3 + 0.9
Standard solutions Pyruvate (11.3) Glucose (282) Glycerol ( 11.8) L-Alanine (56.9) L-Glutamate (56.3) Glycine (57.5) L-Set-me (56.0) Note.
LDHtreated
AMONG
(Bq)
Recovered in ethanol fraction U-W Samples (put on column, nmol)
MATERIALS
values
382 2348 426 414 415 414 414 shown
51.6 2305 419 410 405 410 406 were the means
50.6 2312 418 410 407 410 406
329.9 32.3 7.7 5.1 8.8 4.3 6.4
of duplicate
determinations.
mean standard deviation of the measurements (n = 5 set) was 1.3% of the mean values measured, thus indicating a high degree of repeatability in the process described, in the same range of repeatability as the actual cold lactate spectrophotometric enzymatic measurement ( 1.4%). The actual recovery of other labeled materials is also shown in Table 1. They were tested at concentrations comparable to those found in the rat’s biological fluids and were the most commonly plausible
331.0 30.0 8.0 4.6 8.0 4.4 5.6
1.1 2.3 -0.3 0.5 0.8 -0.1 0.9
0.3 0.1 -0. I 0.1 0.2 -0. I 0.2
contaminants that could interfere in experiments in which labeled lactate is thought to be present (12,13,15,16). No interferences from glucose, pyruvate, or the amino acids tested were observed. Actually, only minor interferences could be expected because the procedure described ensures a high degree of specificity at the enzymatic level and eliminates other interferences through the use of its own blank. This method shows high repetitiveness, is
ENZYMATIC
LACTATE-SPECIFIC
relatively inexpensive, and is applicable to large numbers of samples for the determination of lactate-specific radioactivity in tracer metabolic studies. REFERENCES 1. Katz, J., Okajima, F., Chenoweth, M., and Dunn, A. (1981) Biochem. J. 194, 513-524. 2. Leichtweib, H. P., and Schroder, H. (1981) Pflugers Arch.
390, 80-85.
3. Bernstein, I. A., and Woot, H. G. (1957) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 4, pp. 561-584. Academic Press, New York. 4. Eldridge, F. L., T’so, L., and Chang, H. (1974) J. Appl. Physiol.
37, 3 16-320.
5. Roseman, J. (1953) Amer. Chem. Sot. 75, 3854. 6. Jorfeldt, L. (1970) AC?Q Physiol. Scund. (Suppl) 338, 1.
RADIOACTIVITY
193
DETERMINATION
7. Kusaka, N., and Vi, M. (1973) Anal. Biochem. 52, 369. 8. Relly, P. E. B. (1975) Anal. Biochem. 64, 37-44. 9. Annisol, E. F., Lindsay, D. B., and White, R. R. (1963)
Biochem.
J. 88, 243.
10. Long, C. L., Mashima, J., and Gump, F. (1971) Anal.
Biochem.
40, 386.
11. Dressier, M. (1979) J. Chromatogr. 165, 167-207. 12. Gianotti, M., Rota, P., and Palou, A. (1984) J. Biochem. Biophys. Methods 10, 181-185. 13. Rota, P., Palou, A., and Alemany, M. (1983) J. Biochem.
Biophys.
Methods
8, 63-67.
14. Gutmann, I., and Wahlefeld, A. W. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H. U., ed.), Vol. 3, pp. 1464-1468, Academic Press, New York. 15. Alemany, M., Palou, A., Codina, J., and Herrera, E. (1978) Diabete Metab. 4, 181-186. 16. Okajima, F., Chenoweth, M., Rognstad, R., and Dunn, A. (1981) Biochem. J. 194, 525-540.