ANALYTICAL BIOCHEMISTRY 64, 3 7 - 4 4 (1975)
Use of Reverse Isotope Dilution Analysis to Determine Blood Plasma /(--I-)-14C Lactate Specific Radioactivity P. E. B. REILLY Department of Biochemistry, The University of Liverpool, P.O. Box 147, Liverpool L69 3BX, England R e c e i v e d May 29, 1974; accepted A u g u s t 5, 1974 A method for estimation of the specific radioactivity of blood p l a s m a L ( - ~ - ) - 1 4 C lactate is described. It is based on the l e v e r s e isotope dilution principle and involves a max imum error on any individual sample of 4%. The mean error for a number of determinations is reduced to 0.8%. Duplicate specific ra di oa c t i vi t y values can co nveniently be obtained on six blood samples by one w o r k e r in a normal 8 hr working day.
Several methods have been used for m e a s u r e m e n t of the specific radioactivity of L-lactate in biological materials containing other radiolabeled compounds. T h o s e developed by Kreisberg et al. (1), Riley (2), Hoskins and Patterson (3), and by Schumer et al_ (4), rely only on separation of L-lactate from some other radiolabeled metabolites_ T h e s e methods are all open to the acknowledged criticism that an unknown proportion of the m e a s u r e d radioactivity which is taken to be due to L-lactate may be due to the presence of other unknown labeled metabolites. T h e r e are two methods, those by Jorfeldt (5), and K u s a k a and Ui (6) which are based on incubation of L-lactate with lactate dehydrogenase and N A D , in the presence of carbonyl trapping agents. T h e p y r u v a t e derivatives produced are then assayed for radioactivity_ T h e yields of derivative are variable and repeated assays are required so that a mean proportional recovery of radioactivity, initially present as L-lactate, may be obtained for the whole procedure. Calculations of specific radioactivities of unknowns involve the use of this value. Such reliance on the repeatable attainment of an approximate value is a marked weakness of these two methods. T w o further methods, those developed by Annison, Lindsay, and White (7), and by Long, Mashima, and G u m p (8), both m a k e use of the reverse isotope dilution principle and hence are capable of yielding accurate results. Both h o w e v e r suffer from drawbacks; the f o r m e r is rather protracted (approx 48 hr per sample) and s o m e w h a t involved, whereas 37 Copyright © 1975 by Academic Press, Inc. Printed in the United States. All rights of reproductionin any form reserved.
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P. E. B. REILLY
the latter results in loss of carbon 1 of lactate in an oxidation step which produces acetaldehyde. This would lead to errors in specific radioactivity value in unknown samples if the lactate present were not uniformly labeled. As a result of this assessment of the merits and demerits of these three types of method an improved procedure for blood plasma L(+)-14C-lac tate specific radioactivity determination was developed. It involves: (1) Enzymic measurement of plasma L-lactate concentration. (2) Addition of carrier L-lactate to plasma and its separation from pyruvate. (3) Preparation of pyruvate phenyl hydrazone from the isolated L-lactate. (4) Purification and assay for radioactivity of this derivative. The accuracy of the method was unaffected by a variety of metabolites which were considered possibly to cause complications.
MATERIALS
Glycine buffer I. 0.5 M Glycine, 0.4 M hydrazine hydrate, adjusted to pH 9.0 with 2 M N a O H . Glycine b,ffer 1I. 2.0 M Glycine, 0.8 M hydrazine hydrate, 0.027 M E D T A , adjusted to pH 10.0 with concentrated N a O H . Nicotinamide adenine dinucleotide ( B D H ) 0.03 M solution in deionised water. Standard normal L-lactic acid solution (Boehringer Diluted for use 1 : 2000 with 0.6 M perchloric acid (PCA). Lithium e (+) lactate (Sigma). Aqueous carrier e-lactate solution containing approximately 10 mg L-lactate ion in 0.5 ml. (A 0.1 ml aliquot of this solution diluted to 50 ml with 0.6 M P C A is assayed for L-lactate content when a new batch of the salt is used.) Phenyl hydrazine hydrochloride (BDH) 4% w/v aqueous solution. Sodium acetate ( B D H ) 1 M in deionized water. Sodium chloride ( B D H ) 1 M in deionized water. Formic acid ( B D H ) 0.5 M in deionized water. Dowex 1 ion exchange resin 8% cross linked 100-200 mesh. Lactate dehydrogenase suspension (Boehringer, 400 units/rag protein, 5 mg protein/ml. Scintillation fluid; Toluene and Triton X 100 mixed in ratio 2:1 and containing 5 g P P O and 0.5 g POPOP/litre. [1-14C] sodium pyruvate (Radiochemical Centre, Amersham). A stock solution stored at - 1 5 ° C was prepared containing 50 /zCi and 1 mg sodium pyruvate made to 100 ml with 0.6 M PCA. Further dilutions in 0.6 M P C A containing between 4 and I0 nCi and 30 /zg sodium pyruvate/0.1 ml were prepared as required.
L-LACTATE SPECIFIC RADIOACTIVITY
39
[U-t4C] sodium L-lactate (Radiochemical Centre, A m e r s h a m ) . A stock solution was prepared containing 50 ~,Ci and 1 mg Li L-lactate made to 25 ml with deionized water and stored a t - 15°C. Aliquots diluted with deionized water and with the carrier Li L-lactate solution, containing between 16 and 80 nCi/ml and approximately 10 mg L-lactate/ml were prepared as required. (The L-lactate content of each batch was enzymically determined before use.)
METHODS
A. Enzymic" Determination of L-Lactate Concentration T o 3.0 ml of glycine buffer I is added 0.3 ml of either (a) 0.6 M P C A , (b) diluted normal L-lactic acid standard, (c) diluted Li L-lactate "carrier" solution, (d) deproteinized supernatant prepared from plasma. This latter was obtained by mixing 0.5 ml plasma with 1.0 ml 0.6 M P C A and centrifuging for 5 min at 1500 rpm in a bench centrifuge. To each tube was then added 0.3 ml of 0_03 M N A D solution and 0.02 ml L D H suspension. After mixing they were incubated at 36°C for 10 min in a water bath and then read at 340 nm in glass cells, 1 cm light path in a Gilford model 240 single beam spectrophotometer. Since the molar extinction coefficient of reduced N A D under these circumstances is 6220 the expected extinction for the standard lactic acid solution is 0_257. Results obtained were always within the range 0.250-0.261 thus verifying the accuracy of the method. Batches of the Li L-lactate were found to vary in their L-lactate content and carrier solutions made from new batches of the salt were routinely assayed before use so that maximum accuracy in specific radioactivity determination could be achieved.
B. Separation of Plasma Lactate from Pyruvate Acetate form ion exchange resin columns 3.5 cm long and 0_6 cm diameter were prepared by washing the resin bed successively with 6 ml 1.0 M sodium acetate solution and 4 ml deionized water. P l a s m a lactate and pyruvate were separated using this preparation according to the following scheme. T o 0.5 ml of untreated plasma conveniently contained in a scintillation vial is added 0.5 ml carrier lactate solution and 1.0 ml deionized water. After mixing, the solution is run through a resin column. T h e container is rinsed successively with two 1 ml aliquots of deionized water which are also run through the column. T h e resin bed is then washed with 2 ml of water followed by 2 ml of 0.5 M formic acid. T h e s e four pooled effluents are discarded and the eluate containing the lactic acid is collected in a 10 ml conical b o t t o m centrifuge tube by run-
40
P.
E.
B. R E I L L Y
ning a further 2 ml aliquot of 0.5 M formic acid through the resin. Pyruvate remains bound to the column and can be eluted with a subsequent 2 ml wash of 1 M sodium chloride solution. The effectiveness of the separation was demonstrated by adding aliquots of [ 1-14C] sodium pyruvate or [U-a4C] sodium L-lactate to the plasma/carrier lactate/water mixture described above and collecting the 2 ml fractions individually in scintillation vials which were assayed for radioactivity after addition of 10 ml of scintillation fluid. The percentage of recoveries of activity in the "lactate fraction" when [ 1-14C] pyruvate was added varied from 0 . 1 5 -+- 0.04 (9) to 0.39-+ 0.14 (5) and when [U-14C] lactate was added the range was 9 5 . 7 5 - + 0 . 4 7 (8) to 97.67 -+ 0.20 (5). The mean percentage of recovery of [1-~4C] sodium pyruvate obtained in the final 2 ml chloride wash was 97.15 -+ 0.9 (15). It was found that glucose is quantitatively recovered in the first three effluent fractions described above. This was shown using the glucose oxidase/peroxidase test according to Krebs et al. (9) on PCA deproteinized aliquots prepared from these three pooled effluent fractions, and on an aliquot of deproteinized supernatant from the plasma used. The recovery of glucose in these pooled effluents from six columns was 98.6_+ 0.004%. This was a fortuitous finding since glucose has been shown not to yield a derivative under the condition to be described in the next section, and so does not affect the accuracy of determination of L-lactate specific radioactivity. If plasma glucose specific radioactivity were to be required (as is usually the case when L-lactate specific activity is being investigated) it could very conveniently be obtained after pooling and deproteinization of these three effluent fractions.
C. Preparation of Pyruvate Phenyl Hydrazone from L-Lactate The method was initially tested using 0.5 ml of an aqueous solution of carrier L-lactate (11.16 mg) containing 40 nCi [U-~4C] sodium L-lactate, by adding to this mixture 3 ml glycine buffer lI, 100 mg N A D , and 0.04 ml L D H suspension, and incubating this for 90 min at 36°C. The pH was then adjusted to 2.8-3.0 with concentrated HC1 and the tubes centrifuged for 10 min at 3000 rpm using a bench centrifuge to leave a protein free supernatant which is poured off into a 30 ml boiling tube. To this was then added 1 ml of phenyl hydrazine HC1 solution. Gentle agitation at room temperature for 5 min resulted in crystal formation. Agitation for a further 15 min at room temperature followed by haft an hour standing in ice completed the crystallisation process. The crystals were retained on Millipore filters ( H A W P 02500 H A 0.45 p~m) with gentle suction using a water vacuum pump and washed with 10 ml ice cold deionized water. T h e y were then washed off the filter disc with a jet of
L-LACTATE SPECIFIC RADIOACTIVITY
41
deionized water into a clean 30 ml boiling tube and suspended in 10 ml deionized water, in which they were dissolved by gently boiling for a few moments. The pale yellow solution was allowed to cool to r o o m temperature whereupon after a moment's gentle agitation, fine needle-like yellow crystals started to form. The tubes were then allowed to stand in ice water for 15 min by which time recrystallization w a s complete. They were then harvested on a Millipore filter disc, w a s h e d quickly with 10 ml ice cold deionized water, and dried in a warm air o v e n to constant weight. T h e discs of crystals were then weighed (using a Mettler M5 microbalance) in preweighed scintillation vials and taken up in solution with 1 ml methanol, in which they are very readily soluble. T e n milliliters of scintillator fluid was then added and after 2 hr of dark adaptation they were counted in a B e c k m a n LS 200B liquid scintillation spectrometer. Slight quenching occurs and since there was s o m e variation in the weights of the crystals, quench correction was applied, h o w e v e r counting efficiencies always lay in the range 7 8 - 8 4 % . Table 1 gives the results of ten such analyses. The theoretical specific activity is obtained from the consideration that 1 mole lactate reacts to produce 1 mole of pyruvate which in turn yields 1 mole of pyruvate phenyl hydrazone and hence a theoretical yield o f pyruvate phenyl hydrazone is related to the weight of lactate taken in the ratio 178 to 89. The purity of the derivative was ascertained by preparing it from the aqueous carrier Li L-lactate solution. The derivate gave a melting point of 191-192°C which agrees with that quoted in reference works and ele-
TABLE
I
SPECIFIC ACTIVITIES OF L-LACTATE OBTAINED USING 40 nCi I U-t4C] SODIUM L-LACTATE IN AQUEOUS SOLUTION WITH 11.16 mg CARRIER L-LACTATE. THEORETICAL sp act = 3.58 nCi/mg LACTATE, Weight of
derivative
Equivalent weight of lactate
Activity found
radioactivity
(mg)
(rag)
(nCi)
(nCi/mg)
% Error
8.13 8.82 9.73 9.06 7.63 7,60 5.38 4.87 6.26 8.64
4,06 4.41 4.86 4.53 3.82 3.80 2.69 2.43 3,13 4,32
14.30 16.18 16.95 16.64 13.64 13.38 10.03 8.93 11.38 15.20
3.52 3,67 3,48 3.67 3.57 3.52 3.73 3,67 3.64 3.52 3.59 --+ 0.03 (It))
--1.7 +2.5 --2.8 +2.5 --0.3 1.6 +4,1 +2.5 +1.7 1.7
Mean _+ SEM (n)
Specific
42
P. E. B. REILLY
mental analysis (C, H, and N) yielded values which gave the correct empirical formula (CgH10N~O0. Mass spectrometry gave a mass ion of 178 and N M R showed the correct ratio for protons on the aromatic ring to those on the methyl carbon and the correct positions for these peaks against a reference peak for T M S (deuterated methanol used as solvent to obviate obscuring the methyl group peak). Infrared spectroscopy (using a K B r disc preparation) showed peaks for a nitrogen hydrogen bond and carbon nitrogen vibrations of an aromatic secondary amine together with stretching of a free carboxyl group and carbon hydrogen out of plane deformation of an aromatic ring with five free hydrogens. Characteristic aromatic bands at 1600, 1525, and 1450 cm -1 were also present. The accuracy of the method was also evaluated by determining the specific radioactivity of lactate after its separation from plasma. The details are given in Table 2 which shows the results of eight individual separations and specific radioactivity determinations. For calculation of the specific activity of L(+)-14C-lactate in a 0.5 ml plasma sample obtained during a tracer experiment the following expression is used: 100 × net cpm × 2 × mg lactate initially present for extraction by resin % counting efficiency x 2220 × m g derivative × m g lactate in 0.5 ml plasma giving the answer as nCi/mg plasma lactate. TABLE 2 SPECIFIC ACTIVITIES OF L-LACTATE OBTAINED AFTER EXTRACTION OF L-LACTATE FROM 0.5 rnl PLASMA. 40 nCi [ U - ~ C ] SODIUM L-LACTATE AND 11.16 nag LACTATE CARRIER WERE ADDED AND SINCE THE PLASMA LACTATE CONCENTRATION = 0.231 nag/rnl, TOTAL LACTATE PRESENT AMOUNTED TO 11.275 nag. THEORETICAL sp act = 3.55 nCi/mg LACTATE
Mean -+ SEM (hi
Weight of derivative (mg)
Equivalent weight lactate (rag)
Activity found (nCi)
Specific radioactivity (nCi/mg)
11.51 10.16 8.44 8.36 6.91 6.30 8.30 9.63
5.76 5.08 4.22 4.18 3.45 3.15 4.15 4.81
20.08 18.56 15.30 15.10 12.25 11.29 14.82 17.19
3.49 3.65 3.63 3.61 3.55 3.58 3.57 3.57 3.58 ± 0.02 (8)
% Error --1.7 +2.8 +2.2 +1.7 0 +0.8 +0.5 +0.5
L-LACTATE SPECIFIC RADIOACTIVITY
43
Although the accuracy of the method is not affected by normal concentrations of metabolites in plasma (as shown by the similarity of the results in the fourth columns of Tables 1 and 2), consideration was given to the possibility that increased concentrations of some of them found in various physiological and pathological states, may have an effect. A mean specific radioactivity o f 3.58 + _ 0.01 (8) nCi/mg was obtained under the same conditions as are described in Table 2 except that 15/zg each of oxaloacetic acid, disodium oxoglutarate, and lithium acetoacerate, together with 5 0 / z g sodium pyruvate had been added to the plasma before isolation of L-lactate. This experiment showed that these increases in the amounts of these metabolites in plasma had no effect on the accuracy of the method. Lactate may also be separated from deproteinized filtrates which do not contain anions of the deproteinization reagent. T h e following procedure was found to be suitable using plasma and may be considered to be potentially of use for biological materials which require preliminary deproteinization. T o 0.5 ml plasma (0.18 mg k-lactate/ml) contained in a conical bottomed bench centrifuge tube was added 1.0 ml carrier lactate solution containing 40 nCi [U-14C] and 10.4 mg k-lactate. This gives a theoretical specific activity value of 3.816 nCi/mg L-lactate. T o this plasma/carrier lactate mixture was added 0.5 ml deionized water and 0.1 ml concentrated perchloric acid solution (9.5 M)- After thoroughly mixing and standing at room temperature for 5 rain the tubes were centrifuged at 1000g for 10 rain and the clear supernatant decanted into a second 10 ml conical bottomed centrifuge tube. T h e excess perchloric acid was neutralized by the addition of 0_7 ml of an aqueous 1 M potassium hydroxide solution to a final pH of 6-6.5 and after standing in ice for 5 rain the tubes were centrifuged as described a b o v e to leave the supernatant free from potassium perchlorate. Lactate was separated from the supernatant by elution through the acetate form D o w e x 1 resin column preparation described previously, and recovered, as before, in the second two ml 0.5 M formic acid fraction. A 2/~1 aliquot of this eluate was taken for L-lactate assay and the remainder used for the preparation of pyruvate phenyl hydrazone. T h e mean percentage of recovery of lactate after ion exchange was 85.3 +- 0,8 (6). Presumably this low value is due to the manipulations involved in deproteinization, however, the mean specific activity obtained was 3.84 + 0.04 (6) nCiimg lactate which agrees well with the theoretical value_
ACKNOWLEDGMENTS I should like to thank Dr. R. Ramage of the Department of Organic Chemistry for helpful discussions and Mr. D. Newman of that department for the elemental analyses.
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P. E. B. REILLY
The excellent technical assistance given by Mr. Julian Warner and the help in analysis of the infrared spectroscopy results given by Dr. J. F. Pennock of this department are gratefully acknowledged_
REFERENCES 1. 2. 3. 4. 5, 6. 7. 8. 9.
KREISBERG, R. A., PENNINGTON, k. F_, AND BOSHELL, B. R. (1970) Diabetes 19, 53. RILEY, M. V. (1968)Anal, Biochem. 22, 341. HOSKINS, D. D., AND PATTERSON, D. L. (1968)J. Reprod. Fertil. 16, 183. SCHUMER, W., Moss, G. S_, AND NYHUS, L. M. (1969) Amer. J. Surg. 118, 200. JORFELDT, L. (1970)Acta Physiol. Scand_ Suppl. 338, 1. KUSAKA, M., AND UI, M_ (1973) Anal_ Biochem. 52, 369. ANNISON, E. F., LINDSAY, O. B., AND WHITE, R. R. (1963) Biochem. J. 88, 243. LONG, C. L., MASHIMA, Y., AND GUMP, F_ (1971) Anal. Biochem. 40, 386. KREBS, H. A., BENNETT, D. A. H., DE GASQUET, P., GASCOYNE, T., AND YOSHIDA, T. (1963) Biochem. J, 86, 22.
Use of Reverse Isotope Dilution Analysis to Determine Blood Plasma /(--I-)-14C Lactate Specific Radioactivity P. E. B. REILLY Department of Biochemistry, The University of Liverpool, P.O. Box 147, Liverpool L69 3BX, England R e c e i v e d May 29, 1974; accepted A u g u s t 5, 1974 A method for estimation of the specific radioactivity of blood p l a s m a L ( - ~ - ) - 1 4 C lactate is described. It is based on the l e v e r s e isotope dilution principle and involves a max imum error on any individual sample of 4%. The mean error for a number of determinations is reduced to 0.8%. Duplicate specific ra di oa c t i vi t y values can co nveniently be obtained on six blood samples by one w o r k e r in a normal 8 hr working day.
Several methods have been used for m e a s u r e m e n t of the specific radioactivity of L-lactate in biological materials containing other radiolabeled compounds. T h o s e developed by Kreisberg et al. (1), Riley (2), Hoskins and Patterson (3), and by Schumer et al_ (4), rely only on separation of L-lactate from some other radiolabeled metabolites_ T h e s e methods are all open to the acknowledged criticism that an unknown proportion of the m e a s u r e d radioactivity which is taken to be due to L-lactate may be due to the presence of other unknown labeled metabolites. T h e r e are two methods, those by Jorfeldt (5), and K u s a k a and Ui (6) which are based on incubation of L-lactate with lactate dehydrogenase and N A D , in the presence of carbonyl trapping agents. T h e p y r u v a t e derivatives produced are then assayed for radioactivity_ T h e yields of derivative are variable and repeated assays are required so that a mean proportional recovery of radioactivity, initially present as L-lactate, may be obtained for the whole procedure. Calculations of specific radioactivities of unknowns involve the use of this value. Such reliance on the repeatable attainment of an approximate value is a marked weakness of these two methods. T w o further methods, those developed by Annison, Lindsay, and White (7), and by Long, Mashima, and G u m p (8), both m a k e use of the reverse isotope dilution principle and hence are capable of yielding accurate results. Both h o w e v e r suffer from drawbacks; the f o r m e r is rather protracted (approx 48 hr per sample) and s o m e w h a t involved, whereas 37 Copyright © 1975 by Academic Press, Inc. Printed in the United States. All rights of reproductionin any form reserved.
38
P. E. B. REILLY
the latter results in loss of carbon 1 of lactate in an oxidation step which produces acetaldehyde. This would lead to errors in specific radioactivity value in unknown samples if the lactate present were not uniformly labeled. As a result of this assessment of the merits and demerits of these three types of method an improved procedure for blood plasma L(+)-14C-lac tate specific radioactivity determination was developed. It involves: (1) Enzymic measurement of plasma L-lactate concentration. (2) Addition of carrier L-lactate to plasma and its separation from pyruvate. (3) Preparation of pyruvate phenyl hydrazone from the isolated L-lactate. (4) Purification and assay for radioactivity of this derivative. The accuracy of the method was unaffected by a variety of metabolites which were considered possibly to cause complications.
MATERIALS
Glycine buffer I. 0.5 M Glycine, 0.4 M hydrazine hydrate, adjusted to pH 9.0 with 2 M N a O H . Glycine b,ffer 1I. 2.0 M Glycine, 0.8 M hydrazine hydrate, 0.027 M E D T A , adjusted to pH 10.0 with concentrated N a O H . Nicotinamide adenine dinucleotide ( B D H ) 0.03 M solution in deionised water. Standard normal L-lactic acid solution (Boehringer Diluted for use 1 : 2000 with 0.6 M perchloric acid (PCA). Lithium e (+) lactate (Sigma). Aqueous carrier e-lactate solution containing approximately 10 mg L-lactate ion in 0.5 ml. (A 0.1 ml aliquot of this solution diluted to 50 ml with 0.6 M P C A is assayed for L-lactate content when a new batch of the salt is used.) Phenyl hydrazine hydrochloride (BDH) 4% w/v aqueous solution. Sodium acetate ( B D H ) 1 M in deionized water. Sodium chloride ( B D H ) 1 M in deionized water. Formic acid ( B D H ) 0.5 M in deionized water. Dowex 1 ion exchange resin 8% cross linked 100-200 mesh. Lactate dehydrogenase suspension (Boehringer, 400 units/rag protein, 5 mg protein/ml. Scintillation fluid; Toluene and Triton X 100 mixed in ratio 2:1 and containing 5 g P P O and 0.5 g POPOP/litre. [1-14C] sodium pyruvate (Radiochemical Centre, Amersham). A stock solution stored at - 1 5 ° C was prepared containing 50 /zCi and 1 mg sodium pyruvate made to 100 ml with 0.6 M PCA. Further dilutions in 0.6 M P C A containing between 4 and I0 nCi and 30 /zg sodium pyruvate/0.1 ml were prepared as required.
L-LACTATE SPECIFIC RADIOACTIVITY
39
[U-t4C] sodium L-lactate (Radiochemical Centre, A m e r s h a m ) . A stock solution was prepared containing 50 ~,Ci and 1 mg Li L-lactate made to 25 ml with deionized water and stored a t - 15°C. Aliquots diluted with deionized water and with the carrier Li L-lactate solution, containing between 16 and 80 nCi/ml and approximately 10 mg L-lactate/ml were prepared as required. (The L-lactate content of each batch was enzymically determined before use.)
METHODS
A. Enzymic" Determination of L-Lactate Concentration T o 3.0 ml of glycine buffer I is added 0.3 ml of either (a) 0.6 M P C A , (b) diluted normal L-lactic acid standard, (c) diluted Li L-lactate "carrier" solution, (d) deproteinized supernatant prepared from plasma. This latter was obtained by mixing 0.5 ml plasma with 1.0 ml 0.6 M P C A and centrifuging for 5 min at 1500 rpm in a bench centrifuge. To each tube was then added 0.3 ml of 0_03 M N A D solution and 0.02 ml L D H suspension. After mixing they were incubated at 36°C for 10 min in a water bath and then read at 340 nm in glass cells, 1 cm light path in a Gilford model 240 single beam spectrophotometer. Since the molar extinction coefficient of reduced N A D under these circumstances is 6220 the expected extinction for the standard lactic acid solution is 0_257. Results obtained were always within the range 0.250-0.261 thus verifying the accuracy of the method. Batches of the Li L-lactate were found to vary in their L-lactate content and carrier solutions made from new batches of the salt were routinely assayed before use so that maximum accuracy in specific radioactivity determination could be achieved.
B. Separation of Plasma Lactate from Pyruvate Acetate form ion exchange resin columns 3.5 cm long and 0_6 cm diameter were prepared by washing the resin bed successively with 6 ml 1.0 M sodium acetate solution and 4 ml deionized water. P l a s m a lactate and pyruvate were separated using this preparation according to the following scheme. T o 0.5 ml of untreated plasma conveniently contained in a scintillation vial is added 0.5 ml carrier lactate solution and 1.0 ml deionized water. After mixing, the solution is run through a resin column. T h e container is rinsed successively with two 1 ml aliquots of deionized water which are also run through the column. T h e resin bed is then washed with 2 ml of water followed by 2 ml of 0.5 M formic acid. T h e s e four pooled effluents are discarded and the eluate containing the lactic acid is collected in a 10 ml conical b o t t o m centrifuge tube by run-
40
P.
E.
B. R E I L L Y
ning a further 2 ml aliquot of 0.5 M formic acid through the resin. Pyruvate remains bound to the column and can be eluted with a subsequent 2 ml wash of 1 M sodium chloride solution. The effectiveness of the separation was demonstrated by adding aliquots of [ 1-14C] sodium pyruvate or [U-a4C] sodium L-lactate to the plasma/carrier lactate/water mixture described above and collecting the 2 ml fractions individually in scintillation vials which were assayed for radioactivity after addition of 10 ml of scintillation fluid. The percentage of recoveries of activity in the "lactate fraction" when [ 1-14C] pyruvate was added varied from 0 . 1 5 -+- 0.04 (9) to 0.39-+ 0.14 (5) and when [U-14C] lactate was added the range was 9 5 . 7 5 - + 0 . 4 7 (8) to 97.67 -+ 0.20 (5). The mean percentage of recovery of [1-~4C] sodium pyruvate obtained in the final 2 ml chloride wash was 97.15 -+ 0.9 (15). It was found that glucose is quantitatively recovered in the first three effluent fractions described above. This was shown using the glucose oxidase/peroxidase test according to Krebs et al. (9) on PCA deproteinized aliquots prepared from these three pooled effluent fractions, and on an aliquot of deproteinized supernatant from the plasma used. The recovery of glucose in these pooled effluents from six columns was 98.6_+ 0.004%. This was a fortuitous finding since glucose has been shown not to yield a derivative under the condition to be described in the next section, and so does not affect the accuracy of determination of L-lactate specific radioactivity. If plasma glucose specific radioactivity were to be required (as is usually the case when L-lactate specific activity is being investigated) it could very conveniently be obtained after pooling and deproteinization of these three effluent fractions.
C. Preparation of Pyruvate Phenyl Hydrazone from L-Lactate The method was initially tested using 0.5 ml of an aqueous solution of carrier L-lactate (11.16 mg) containing 40 nCi [U-~4C] sodium L-lactate, by adding to this mixture 3 ml glycine buffer lI, 100 mg N A D , and 0.04 ml L D H suspension, and incubating this for 90 min at 36°C. The pH was then adjusted to 2.8-3.0 with concentrated HC1 and the tubes centrifuged for 10 min at 3000 rpm using a bench centrifuge to leave a protein free supernatant which is poured off into a 30 ml boiling tube. To this was then added 1 ml of phenyl hydrazine HC1 solution. Gentle agitation at room temperature for 5 min resulted in crystal formation. Agitation for a further 15 min at room temperature followed by haft an hour standing in ice completed the crystallisation process. The crystals were retained on Millipore filters ( H A W P 02500 H A 0.45 p~m) with gentle suction using a water vacuum pump and washed with 10 ml ice cold deionized water. T h e y were then washed off the filter disc with a jet of
L-LACTATE SPECIFIC RADIOACTIVITY
41
deionized water into a clean 30 ml boiling tube and suspended in 10 ml deionized water, in which they were dissolved by gently boiling for a few moments. The pale yellow solution was allowed to cool to r o o m temperature whereupon after a moment's gentle agitation, fine needle-like yellow crystals started to form. The tubes were then allowed to stand in ice water for 15 min by which time recrystallization w a s complete. They were then harvested on a Millipore filter disc, w a s h e d quickly with 10 ml ice cold deionized water, and dried in a warm air o v e n to constant weight. T h e discs of crystals were then weighed (using a Mettler M5 microbalance) in preweighed scintillation vials and taken up in solution with 1 ml methanol, in which they are very readily soluble. T e n milliliters of scintillator fluid was then added and after 2 hr of dark adaptation they were counted in a B e c k m a n LS 200B liquid scintillation spectrometer. Slight quenching occurs and since there was s o m e variation in the weights of the crystals, quench correction was applied, h o w e v e r counting efficiencies always lay in the range 7 8 - 8 4 % . Table 1 gives the results of ten such analyses. The theoretical specific activity is obtained from the consideration that 1 mole lactate reacts to produce 1 mole of pyruvate which in turn yields 1 mole of pyruvate phenyl hydrazone and hence a theoretical yield o f pyruvate phenyl hydrazone is related to the weight of lactate taken in the ratio 178 to 89. The purity of the derivative was ascertained by preparing it from the aqueous carrier Li L-lactate solution. The derivate gave a melting point of 191-192°C which agrees with that quoted in reference works and ele-
TABLE
I
SPECIFIC ACTIVITIES OF L-LACTATE OBTAINED USING 40 nCi I U-t4C] SODIUM L-LACTATE IN AQUEOUS SOLUTION WITH 11.16 mg CARRIER L-LACTATE. THEORETICAL sp act = 3.58 nCi/mg LACTATE, Weight of
derivative
Equivalent weight of lactate
Activity found
radioactivity
(mg)
(rag)
(nCi)
(nCi/mg)
% Error
8.13 8.82 9.73 9.06 7.63 7,60 5.38 4.87 6.26 8.64
4,06 4.41 4.86 4.53 3.82 3.80 2.69 2.43 3,13 4,32
14.30 16.18 16.95 16.64 13.64 13.38 10.03 8.93 11.38 15.20
3.52 3,67 3,48 3.67 3.57 3.52 3.73 3,67 3.64 3.52 3.59 --+ 0.03 (It))
--1.7 +2.5 --2.8 +2.5 --0.3 1.6 +4,1 +2.5 +1.7 1.7
Mean _+ SEM (n)
Specific
42
P. E. B. REILLY
mental analysis (C, H, and N) yielded values which gave the correct empirical formula (CgH10N~O0. Mass spectrometry gave a mass ion of 178 and N M R showed the correct ratio for protons on the aromatic ring to those on the methyl carbon and the correct positions for these peaks against a reference peak for T M S (deuterated methanol used as solvent to obviate obscuring the methyl group peak). Infrared spectroscopy (using a K B r disc preparation) showed peaks for a nitrogen hydrogen bond and carbon nitrogen vibrations of an aromatic secondary amine together with stretching of a free carboxyl group and carbon hydrogen out of plane deformation of an aromatic ring with five free hydrogens. Characteristic aromatic bands at 1600, 1525, and 1450 cm -1 were also present. The accuracy of the method was also evaluated by determining the specific radioactivity of lactate after its separation from plasma. The details are given in Table 2 which shows the results of eight individual separations and specific radioactivity determinations. For calculation of the specific activity of L(+)-14C-lactate in a 0.5 ml plasma sample obtained during a tracer experiment the following expression is used: 100 × net cpm × 2 × mg lactate initially present for extraction by resin % counting efficiency x 2220 × m g derivative × m g lactate in 0.5 ml plasma giving the answer as nCi/mg plasma lactate. TABLE 2 SPECIFIC ACTIVITIES OF L-LACTATE OBTAINED AFTER EXTRACTION OF L-LACTATE FROM 0.5 rnl PLASMA. 40 nCi [ U - ~ C ] SODIUM L-LACTATE AND 11.16 nag LACTATE CARRIER WERE ADDED AND SINCE THE PLASMA LACTATE CONCENTRATION = 0.231 nag/rnl, TOTAL LACTATE PRESENT AMOUNTED TO 11.275 nag. THEORETICAL sp act = 3.55 nCi/mg LACTATE
Mean -+ SEM (hi
Weight of derivative (mg)
Equivalent weight lactate (rag)
Activity found (nCi)
Specific radioactivity (nCi/mg)
11.51 10.16 8.44 8.36 6.91 6.30 8.30 9.63
5.76 5.08 4.22 4.18 3.45 3.15 4.15 4.81
20.08 18.56 15.30 15.10 12.25 11.29 14.82 17.19
3.49 3.65 3.63 3.61 3.55 3.58 3.57 3.57 3.58 ± 0.02 (8)
% Error --1.7 +2.8 +2.2 +1.7 0 +0.8 +0.5 +0.5
L-LACTATE SPECIFIC RADIOACTIVITY
43
Although the accuracy of the method is not affected by normal concentrations of metabolites in plasma (as shown by the similarity of the results in the fourth columns of Tables 1 and 2), consideration was given to the possibility that increased concentrations of some of them found in various physiological and pathological states, may have an effect. A mean specific radioactivity o f 3.58 + _ 0.01 (8) nCi/mg was obtained under the same conditions as are described in Table 2 except that 15/zg each of oxaloacetic acid, disodium oxoglutarate, and lithium acetoacerate, together with 5 0 / z g sodium pyruvate had been added to the plasma before isolation of L-lactate. This experiment showed that these increases in the amounts of these metabolites in plasma had no effect on the accuracy of the method. Lactate may also be separated from deproteinized filtrates which do not contain anions of the deproteinization reagent. T h e following procedure was found to be suitable using plasma and may be considered to be potentially of use for biological materials which require preliminary deproteinization. T o 0.5 ml plasma (0.18 mg k-lactate/ml) contained in a conical bottomed bench centrifuge tube was added 1.0 ml carrier lactate solution containing 40 nCi [U-14C] and 10.4 mg k-lactate. This gives a theoretical specific activity value of 3.816 nCi/mg L-lactate. T o this plasma/carrier lactate mixture was added 0.5 ml deionized water and 0.1 ml concentrated perchloric acid solution (9.5 M)- After thoroughly mixing and standing at room temperature for 5 rain the tubes were centrifuged at 1000g for 10 rain and the clear supernatant decanted into a second 10 ml conical bottomed centrifuge tube. T h e excess perchloric acid was neutralized by the addition of 0_7 ml of an aqueous 1 M potassium hydroxide solution to a final pH of 6-6.5 and after standing in ice for 5 rain the tubes were centrifuged as described a b o v e to leave the supernatant free from potassium perchlorate. Lactate was separated from the supernatant by elution through the acetate form D o w e x 1 resin column preparation described previously, and recovered, as before, in the second two ml 0.5 M formic acid fraction. A 2/~1 aliquot of this eluate was taken for L-lactate assay and the remainder used for the preparation of pyruvate phenyl hydrazone. T h e mean percentage of recovery of lactate after ion exchange was 85.3 +- 0,8 (6). Presumably this low value is due to the manipulations involved in deproteinization, however, the mean specific activity obtained was 3.84 + 0.04 (6) nCiimg lactate which agrees well with the theoretical value_
ACKNOWLEDGMENTS I should like to thank Dr. R. Ramage of the Department of Organic Chemistry for helpful discussions and Mr. D. Newman of that department for the elemental analyses.
44
P. E. B. REILLY
The excellent technical assistance given by Mr. Julian Warner and the help in analysis of the infrared spectroscopy results given by Dr. J. F. Pennock of this department are gratefully acknowledged_
REFERENCES 1. 2. 3. 4. 5, 6. 7. 8. 9.
KREISBERG, R. A., PENNINGTON, k. F_, AND BOSHELL, B. R. (1970) Diabetes 19, 53. RILEY, M. V. (1968)Anal, Biochem. 22, 341. HOSKINS, D. D., AND PATTERSON, D. L. (1968)J. Reprod. Fertil. 16, 183. SCHUMER, W., Moss, G. S_, AND NYHUS, L. M. (1969) Amer. J. Surg. 118, 200. JORFELDT, L. (1970)Acta Physiol. Scand_ Suppl. 338, 1. KUSAKA, M., AND UI, M_ (1973) Anal_ Biochem. 52, 369. ANNISON, E. F., LINDSAY, O. B., AND WHITE, R. R. (1963) Biochem. J. 88, 243. LONG, C. L., MASHIMA, Y., AND GUMP, F_ (1971) Anal. Biochem. 40, 386. KREBS, H. A., BENNETT, D. A. H., DE GASQUET, P., GASCOYNE, T., AND YOSHIDA, T. (1963) Biochem. J, 86, 22.
