Anal. Chem. 1993, 65,992-993
992
Enzymatic Determination of Carbon- 14-Labeled D-@-Hydroxybutyratein Biological Samples S. Quevedo, A. Palou,' and P. Roca Departament d e Biologia Fonarnental i Cikncies de la S a l u t (Bioquimica i Biologia Molecular), Universitat d e les Illes Balears i Institut d'Estudis Auancats (UIB-CSIC), Ctra Valldemossa K m 7.5, 07071 Palma d e Mallorca, S p a i n
A method for determlnatlon of D-B-hydroxybutyrate-speclflc radloactlvlty In blologlcal samples Is presented. I t Is based on the followlng steps: (a) enzymatlc converslon of D-Bhydroxybutyrateto acetoacetate and Its conversion to phenylhydrazone, (b) quantltatlvetrapplng of the phenylhydrazone In SPE C18 columns, (c) eluatlon wlth ethanol of the phenylhydrazone of acetoacetate followed by radloactlvlty countlng, and (d) estlmatlon of the radloactlvlty thus trapped compared wlth that of enzymatically untreated allquots of the same samples. No Interferences from other i4C-labeled materlalssuch as ~ I u c o s eL-alanlne, , Lgluiamlne, and L-vallne were observed. Thls lnexpenslveand hlgh-speed method can be applied In routlne multiple estlmations of D-8-hydroxybutyratsspeclflc radloactlvlty In blologlcal samples In tracer metabolic studles.
INTRODUCTION Determination of [ I4C]-0-hydroxybutyrate radioactivity is of interest in metabolic studies on fatty acid metabolism or ketone body utilization as a metabolic fuel. T h e study of both the production and t h e utilization of ketone bodies in vivo is tedious, because t h e available methods for 14C-labeled ketone bodies radioactivity quantification are considerably time consuming.1-6 These methods are based on ketone body decarboxylation in t h e presence of acids, followed by acetone collection and or on t h e ability of P-hydroxybutyrate to quantitively bind t o and elute from a n ion-exchange resin.6 T h e use of polymeric porous hydrophobic adsorbing beads to recover hydrophobic compounds from aqueous solutions is a well-documented Also we had previously applied this property to t h e estimation of individual amino acid radioactivity in plasma ~ a m p l e s ammonia ,~ and urea determination in water samples,I2 enzymatic L-lactate radioactivity,ll pyruvic-specific radioactivity,IO and ' C l a b e l e d ~-alanine13in biological samples. ~
~~
* To whom correspondence should be addressed.
(1) Van Slyke, D. D. J. Biol. Chem. 1917, 32, 455. (2) Van Slyke, D. D. J. Biol. Chern. 1929, 82, 415. (3) Mayes, P. A,; Felts, J. M. Biochem. J . 1967, 102, 230. (4) Emmanuel,B.; Stangassinger, M.; Giesecke D. Ann. Clin. Biochern. 1979, 16, 155-160. (5) Mewes, W.; Seufert, C. D.; Soling, H. D. Anal. Biochern. 1979. 92, 394-402. (6) Miles, J. M.; Haymond, M. W.; Rima, R. A.; Gerich. J. E. J . L i p i d Res. 1980, 21, 646-650. (7) "Baker"-10 SPE'" ADolications Guide. Vol. I. J T. Baker Chemil .. Co., 1982. (8) 'Baker"-10 SPErhlApplicationsGuide. Vol. 11. J. T. Baker Chemil Co., 1984. (9) Roca, P.; Palou, A,; Alemsny, M. Biochern. Biophys. Methuds 1983, 8, 63-67. (10) Gianotti,M.;Roca,P.: Palou, A.Biochern.Biophys. Methods 1984, 10, 181-185. (11) Roca, P.; Gianotti, M.; Palou. A. Anal. Chern. 1985,57, 148, 190193. (12) Moreno, P.; Sbnchez, E.; Pons, A,; Palou A. Anal. Chern. 1986,58, 585-587. (13) Serra, F.: Palou, A,: Pons A . .4nai. Chern. 1985. 59, 1841-1843. 0003-2700/93/0365-0992$04.00/0
In this paper we present a method for D-0-hydroxybutyratespecific radioactivity determination based on t h e highly specific enzymatic transformation of D-0-hydroxybutyrate t o acetoacetate catalyzed by t h e enzyme D-6-hydroxybutyrate dehydrogenase (EC 1.1.1.30), coupled t o t h e simultaneous formation of t h e respective phenylhydrazone derivate as follows: CH~CHOHCH~COO +- NAD+
+
QNHNH,
-+
T h e procedure is completed by the quantitative trapping of t h e phenylhydrazone in a simple purification step in SPE C18 columns followed by radioactivity counting of ethanol eluate.
EXPERIMENTAL SECTION Analytical-grade reagents were used. Labeled material was obtained from Radiochemical Center (Amersham, U.K.) and Dupont Nen (Germany). Biochemical standards and reagents were obtained from Sigma Chemical Co. (St. Louis, MO). The SPE C18 columns were obtained from J. T Baker Chemical Co. (Phillipsburg, N J). Samples of rat plasma or standard solutions containing known amounts of potassium, D-(-)-@- [3-'4Cl-hydroxybutyricacid (from 0.25 to 1.5 mM; final specific radioactivity of 5.4 MBqimmol) were used. The solutions of U-14C-labeled material used to validate the method contained 0.60 mM L-alanine (16.1 MBqi mmol), 0.50 mM L-glutamine (20.5 MBqimmol), 6 mM D-glucose (0.9MBqimmol), and0.20mM~-valine(42.2 MBqimmol). These solutions were handled under standard safety regulations for the use of radiochemical materials in laboratories. One milliliter of sample, previously diluted 1:l with distilled water, was deproteinized with 1 mL of 12% (wiv) perchloric acid. Supernatants were then neutralized with 0.35 mL of 2 N KOH containing 2 N KHCO? and centrifuged again. Two aliquots of 0.25 mL from each final supernatant were used. The first, A, was incubated with 1 mL of buffer phenylhydrazine-Tris (0.10 M Tris pH = 8.5 with 2.7 mM EDTANa2and C1H 0.25 N and 1 L c phenylhydrazine) and 0.10 mL of 14 mM NAD+, and excess of P-hydroxybutyrate dehydrogenase (10 pL of 5 unitsiml). The NADH 334 nm extinction increase was measured and used to calculate the D-/3-hydroxybutyrate in samples. The second aliquot, B, was incubated under the same conditions but with n o enzyme. Then both aliquots were processed in parallel. One milliliter of each of the samples was then passed through small 3-mL extraction columns SPE C18 containing 500 mg of sorbent (Phillipsburg, NJ), previously washed with 6 mL ethanol 9 8 5 and equilibrated with 6 mL of water; the columns were recycled several times after 9 8 7 ethanol washing. The samples passed through columns with help of the Baker21 SPE extraction system with vacuum bomb; 1-mL samples were eluted and the column washed with 0.5 mL water. Finally the phenylhydrazone of acetoacetate was eluted with 2 mL of 98'~ ethanol. The 2 mL of eluated ethanol was mixed with 10 mL of liquid scintillation of OptiPhase 'HiSafe' I1 (LKB. UK, 1993 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 65, NO. 8, APRIL 15, 1993
993
Table I. Distribution and Recoveries of [14C]-/3-HydroxybutyrateRadioactivity and Other leC-LabeledMaterials among Different Fractions Obtained with the Method Described. radioactivitv (Rnl
14Csamples (put in column, nmol)
standard solution &hydroxybutyrate (61.5) d-hydroxybutyrate (123) d-hydroxybutyrate (246) fl-hydroxybutyrate (369)
added a
recovered in water fraction enzyme enzyme treated untreated
recovered in ethanol fraction treated, A
untreated, B
enzyme
difference A-B
enzyme
40 f 12 63 11 144 f 29 375 f 5
323 f 2 650 f 5 1340 13 1940 20
298 f 2 579 11 1148 f 22 1642 f 20
9*1 13 f 1 30 f 2 30 2
*
280 3 566 f 11 1124 f 22 1613 22
1815
298 f 40
1790 f 30
1516 f 31
24 f 2
1492 f 30
590 1029 1089
529 f 13 .999*11 1038f8 706 2
536 10 997 16 1048 f 19 707 f 4
5Of1 34f 2 45f2 123 3
48*1 31 f 1 40f 1 127 f 1
*
plasma
fl-hydroxybutyrate (80) standard solutions D-glucose (640) L-alanine (64) L-glutamine (53) L-valine (21) a
*
332 663 1326 1989
886
*
* *
*
2fl 3f2 6f1 4f3
Final recovery (A-B) X 100/a
*
84.3 0.9 85.4 f 1.7 84.8f 1.7 81.1 & 1.5
* 0.37 * 0.22 0.33 * 0.22 0.53 * 0.13 -0.43 * 0.28 82.2 1.7
The values are mean f sem of four determinations.
The differences between both parallel sample aliquots (A and B) gave the D-8-hydroxybutyrateradioactivity. The D-&hydroxybutyrate-specific radioactivity could then be calculated.
RESULTS AND DISCUSSION Table I shows the relation between D-P-hydroxybutyrate radioactivity present in the samples (standard and plasma) in front of radioactivity found in the ethanol eluate with the method described. The steps used in the method can be explained as follows: Firstly the enzymatic conversion of D-8hydroxybutyrate to acetoacetate and its transformation to phenylhydrazone implies the conversion of a hydrophylic to hydrophobic compound. This change in physical properties is highly specific because of the enzymatic characteristics of the transformation.l* The latter steps allow the separation of D-P-hydroxybutyrate carbons from other hydrophylic compounds. The lineal correlation coefficientof added in front of found radioactivity was r = 0.997, n = 16. The mean percentage of recovery of D-P-hydroxybutyrate was 84% (n = 16). No differences were observed between the recoveries from the standard solutions and those from rat plasma (82.2 f 1.7 % ) samples. There was a mean loss of 16% of radioactivity in whole derivatization/purificationthat must be taken into account in the calculation of the originalD-@-hydroxybutyrate (14) Williamson,D.H.; Mellanby,J.InMetho&ofEnzymatic Analysis; Bergmeyer, H. U., Ed.;Academic Press, Inc.: New York, 1974;Vol. 4,pp
1836-1839. (15)Bergmeyer, H. U.;Cawehn, K.; Klotzsch, H.; Krebs, H. A.; Williamson, D. H. Biochern. J. 1967,102,423-427.
radioactivity. This figure is very close to that described in the methods based on the formation of phenylhydrazones,10J1,13 The specificity of this method is the same as the spedrophotometric enzymatic measurement of D-8-hydroxybutyrate.14 With respect to possible interference in the D-0hydroxybutyrate studies from otherp-hydroxy acids or a-keto acids, it is interesting to note that only the higher homologs of D-P-hydroxybutyrate, 3-hydroxypentanoicand 3-hydroxyhexanoic acids, could also react but at much slower rates,16 and these compounds are not present in biological samples. Other possible interferences from non-p-hydroxy acids have been ruled out in the present study. The recovery of the other labeled materials is also shown in Table I. No interferences from D-glucose, L-glutamine, L-valine, and L-alanine were observed using our conditions in concordance with the specificity of P-hydroxybutyrate dehydrogenase for this substrate and ita reactivity with phenylhydrazine. In addition this method is as simple as to be able to be applied to large numbers of samples for the determination of D-P-hydroxybutyrate radioactivity in tracer metabolic studies.
ACKNOWLEDGMENT Work supported by grant PTR89-0053from the "Comisi6n Interministerial de Ciencia y Technologia (CICYT)" and PB89-0426 of the goverment of Spain. RECEIVED for review May 29, 1992. Accepted November 19, 1992.
