ANALYTICAL
BIOCHEMISTRY
67, I’?- 179 ( i 975)
Quantitative and Succinic
Extraction Acids and
by Gas-Liquid C. V. WARNER Department of Biochemistry,
of Methylmalonic Their Determination
Chromatography1 AND GEORGE
V. VAHOUNY
The George Washington University Medical Center, Washington, D.C. 20037
Received October 3, 1974: accepted February 10, 1975 A method for routine small-volume solvent extraction, derivitization and gasliquid chromatographic analysis of methylmalonic and succinic acids is described. The procedure allows for quantitative determination of mass and radioactivity of these acids and can be applied to metabolic studies of the genetic disorder methylmalonylacidurea.
Methods currently available for the extraction and determination of dicarboxylic acids, such as methylmalonic and succinic acids, are generally qualitative and cumbersome for routine application. Isolation of these acids from urine or tissue homogenates has often involved continuous, large-volume ether extractions (l-6) or chloroform and methanolwater extractions (7,8). Subsequent separation of these crude extracts is accomplished by ion-exchange or partition chromatography using large solvent-volumes (1,2,4,8-12), by paper chromatography (1,13,14,15), thin layer silicic acid chromatography (8,16,17), or, more recently, gasliquid chromatography (2,5,9,17- 19). With the exception of gas-liquid chromatography (glc), subsequent analysis of the acids following separation is by alkali titration (10) or, in the case of substituted malonic acids, by spectrophotometry following diazotization with p-nitroaniline (4). Accurate measurements of methylmalonic and succinic acids, as well as several other acids, have been hindered by the volatility of the unionized acids and their derivatives, which results in significant losses during concentration of the large volumes of solvent following extraction and during purification by chromatography. Cardinale et al. (1.5) described a spectrophotometric assay for methylmalonyl coenzyme A which is based on coupled reactions resulting in oxidation of NADH. However, these authors relied on less quantitative extraction and on paper chromatography for determination of isotopic methylmalonate. The problem of variable recoveries of organic acids can, in part, be corI This study was supported by Public Health Service Grant No. HL-09489.
Copyright @ 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
METHYLMALONIC
AND
SUCCINIC
ACIDS
123
rected by addition and recovery of radioactive carriers for each of the acids tested (18), but this precludes the measurement of metabolically derived acids from radioactive precursors. The report of methylmalonic acidurea as an inherited metabolic disorder (20) and the indication that biooxidation of polyunsaturated fatty acids may lead to formation of propionic acid and subsequently to methylmalonic and succinic acids (21,22) have emphasized the need for a more quantitative and relatively routine method for the extraction of these acids and for the quantitative analysis of mass and radioactivity of these acids during metabolic studies. The present report describes a technique for small-volume ether extraction of methylmalonic and succinic acids (as we11 as several other organic acids) which, when combined with gas-liquid radiochromatography following derivatization, permits routine and quantitative analysis of the specific radioactivities of these acids. EXPERIMENTAL
Materials The dicarboxylic acids were obtained from Calbiochem and fatty acids were from The Hormel Institute. [ 1,4-‘-C] Succinic acid was obtained from Amersham/Searle Corp. and [ 2-methyl-14C] malonic acid was purchased from New England Nuclear Corp. All solvents were distilled and reagents were of highest purity. Procedure 1. Extraction of organic acids. Since our studies have been concerned primarily with polyunsaturated fatty acids as potential sources of propionate, methylmalonate and succinate in heart and skeletal muscle, the procedure described here has been applied to in vitro incubation systems containing one or more radioactive compounds. These included linoleic, palmitic, propionic, methylmalonic and succinic acids, as well as certain other dicarboxylic acids described in the text. The incubation media for studies on biooxidation of fatty acids by whole tissue homogenates or mitochondria is described elsewhere (23). Incubations are carried out in capped 25-ml Erlenmeyer flasks containing a polyethylene center well. At the end of the incubation, hyamine hydroxide (0.3 ml) is injected through the rubber cap into the center well, and 0.23 ml of 6 N H&SO, is injected into the incubation medium. Incubation is continued at 37°C for 60 min to allow complete absorption of the evolved [‘“Cl 02, by hyamine, and the center well is removed and placed in 10 ml of scintillation cocktail in a liquid scintillation vial for determination of radioactivity (24).
124
WARNER
AND
VAHOUNY
The contents of the incubation vial are adjusted to pH 12 with NaOH to hydrolyze the CoA thioesters of the organic acids (25) and is then reacidified to pH 3.0 with H&SO+ Solid sodium chloride (ca. 400 mg) is added and the contents of the incubation flask are decanted quantitatively into a screw-cap tube using a total of 5 ml of diethyl ether. The biphasic mixture of ether and water is mixed thoroughly with a tube buzzer, the phases are allowed to separate, and the ether layer is transferred to a second screw-cap tube. The aqueous residue is extracted twice more in the same manner with 5-ml aliquots of ether, and the ether extracts are combined. When fatty acids are absent from the original incubation mixture, 1 mg of stearic acid is added to the ether extract to prevent volatilization losses of the short-chain dicarboxylic acids. Also, at this point, carrier (2 mg) methylmalonic and/or succinic acid is added when appropriate. Excess anhydrous Na,SO, is added to dry the solvent, the tubes are shaken and allowed to stand overnight. The ether extract is decanted into a third tube and the Na.SO, residue is washed twice with 3-5 ml of ether, combining the washings with the extract. The dehydrated extract is carefully evaporated to dryness in an ice bath under nitrogen; during the evaporation, the sides of the tube are frequently rinsed down with ether to reduce drying and evaporation of the acids. 2. Methylation. One milliliter of boron trifluoride-methanol reagent (14% by weight, Applied Science Co.) is added immediately after evaporation of the ether, the tubes are tightly capped and placed in a 70°C tube-heater for 2 hr. The tubes are cooled, 1 ml of distilled water is added, and the methyl esters are extracted with ether by the same procedure used above for extraction of the free un-ionized acids. This is done with three extractions using aliquots of 2 ml of ether twice and 1.5 ml of ether twice. The ether extracts are combined, dried with anhydrous sodium sulfate as before, and quantitatively transferred to calibrated centrifuge tubes. The ether is slowly evaporated to 1.0 ml in an ice-salt bath under nitrogen. Throughout the procedure, where possible, the tubes are maintained in ice baths to avoid volatilization of the methyl ester derivatives of the short-chain mono- and dicarboxylic acids. 3. Gas-liquid radioclzromatography. Gas chromatographic separation was carried out using a Beckman GC-55 gas chromatograph with a stream-splitter (1:20) and flame-ionization detectors. The glass column (6 ft X 0.25 in., o.d.) was packed with 15% HIEFF-2BP (ethylene glycol succinate) on IOO-200-mesh Gas-chrom P (Applied Science Co.). Analysis was carried out at helium carrier gas pressure of 20 psi, and injection block temperature was 250°C. Hydrogen flow was 45 cm3/min, and air flow was 270 cm3/min. Initial oven temperature was maintained at 60°C for 3 min, followed by a programmed temperature increase of
METHYLMALONIC
AND
SUCCINIC
ACIDS
125
7.5”C/min to 19o”C, which was maintained for 14 min for elution of long-chain polyunsaturated fatty acid derivatives. The horizontal arm from the stream-splitter for effluent gas collection passed through a 200°C block-heater (Barber-Coleman Co.) prior to trapping of the sample as described by Howard and Kittinger (26). The effluent gasses passed through a Pasteur pipet containing glass wool which had been treated with liquid scintillant ( 100 mg of 1,4-di-(2,5phenyloxazolyl)benzene and 4 gm of 2,5-diphenyloxazole per liter of toluene) and dried with nitrogen gas. The pipet was replaced at predetermined times during chromatography, based on simultaneous detection and recording of the chromatographic peaks. The contents of the pipet were then eluted into scintillation vials using a total of 10 ml of scintillation fluid. Radioactivity was determined on a three-channel Nuclear Chicago liquid scintillation spectrometer (Mark I). RESULTS
AND
DISCUSSION
Gas chromatography has been successfully applied to the detection of short- and long-chain monocarboxylic acids (e.g., 2,5,9,17-19,27). However, before glc can be applied to biological materials, some degree of purification of the materials is necessary (12). Due to the variable volatility of these shorter-chain acids and their low concentrations in biological materials, the solvent extraction and evaporation procedures generally result in significant losses even before analysis can be achieved (9,18). These losses are further exaggerated prior to gas chromatography after derivatization of the acids to the less polar methyl esters (9). For example, it has been reported that up to 90% of methyl lactate and 70% of trimethyl citrate may be lost by evaporation of a chloroform extract (18). Several techniques have been reported to overcome some of these problems. Stokke et al. (17) and Cardinale et al. (15) have described methods for extraction of methylmalonate from urine or tissue homogenates using small volumes of diethyl ether (9-l 6 ml); reduced losses of volatile acids during evaporation of solvents or during column chromatography have been accomplished by addition of methyl pelargonate. which can also serve as an external standard for glc (2). As mentioned earlier, the addition of radioactive standards to correct for losses throughout extraction and purification procedures has been suggested by Barnett et al. (IS), and Sprinkle et al. (5) have employed the ditrimethylsilyl ester of methylmalonate for glc in order to avoid ionexchange or thin layer chromatography for preliminary purification of ether extracts of urine. The procedure presented in this communication includes: (a) The use of small volumes of diethyl ether for quantitative extraction of methylmalonic and succinic acids from biological materials; (b) the addition of
126
WARNER
AND VAHOUNY
octadecanoic acid to reduce the volatility of the short-chain dicarboxylic acids and their methyl esters during the procedure; (c) the controlled evaporation of diethyl ether solvent containing methyl esters of these acids; and (d) the use of gas-liquid chromatography for quantitative determination and radioactivity of these compounds. This procedure has proven to be a useful routine method for the separate measurements of mass and radioactivity employed previously. The data in Table 1 summarize the recoveries of added labeled methylmalonic and succinic acids from mitochondrial incubation media or tissue homogenates. It is apparent that the undissociated acids (pH 2.0) are quantitatively extracted into small volumes (20 ml) of diethyl ether without the necessity for continuous extraction procedures. For metabolic studies, the procedure initially allows quantitative recovery of [‘“Cl 0, by acidification of the mitochondrial incubation medium to pH 2 and, following adjustment of the medium to pH 12 to hydrolyze the coenzyme A thioester bonds and extraction of the medium at pH 3 with ether, provides quantitative recovery of certain metabolic intermediates. When the incubation medium contained long-chain fatty acids or if carrier octadecanoic acid was added prior to extraction, there were no losses of methylmalonic and succinic acids during evaporation of the iniTABLE RECOVERY
OF METHYLMALONIC DERIVATIZATION
1
AND SUCCINIC AND GAS-LIQUID
ACIDS DURING CHROMATOGRAPHY
EXTRACTIONS,
% Recovery of cL Procedures 1. Ether extraction, pH 2-3 incubation medium 2. Extraction and evaporation of ether extracts a. In absence of carrier octadecanoic acid b. In presence of carrier octadecanoic acid 3. Derivatization, ester extraction, and evaporation of ether 4. Complete procedure (steps 1, 2b. 3) 5. Gas-liquid tography
A Methylmalonic
B Succinic acid
A and B mixture
98 2 1
101 k 1
96 2 5
90 f 1
91 2 1
93 2 3
97
99
91 i 1
88 2 1
91 F 2
93 * 1
85 r 2
88 f 1
100.7 k 2.7
97.9 f 2.0
104
acid
radiochroma-
a The values represent means 2 SEM for up to ten determination.
-
METHYLMALONIC
AND
SUCCINIC
127
ACIDS
tial ether extracts under nitrogen in preparation for derivatization (Table 1, Procedure 2b). However, in the absence of carrier, significant volatilization of these acids occurred, especially during the final stages of evaporation. This could, in part, be prevented by maintaining the extracts in ice during the entire procedure. Derivatization with BF,-methanol and subsequent extraction of methyl esters have been effectively employed earlier for short- and longchain fatty acids and presented no unique problems with methylmalonic and succinic acids (Table 1, Procedure 3). However, evaporation of the ether extracts of the dimethyl esters of these acids was the major source of losses in the entire procedure. These evaporations were carried out in ice, but it was found that significant losses occurred during complete evaporation of the solvent. By carefully and regularly washing down the sides of the tube with small volumes of ether, and preventing the volume of solvent to fall below 1.0 ml, it was possible to avoid major losses of the two short-chain dicarboxylic acids (Table 1, Procedure 4). This final evaporation step is then the major limitation in the procedure in terms of providing a concentrated preparation of derivatized methylmalonic and succinic acids for gas-liquid chromatography. The same limitation is expected with the trimethylsilyl derivatives (5) of these acids. Using a 20: 1 stream-splitter, in which the major column effluent was trapped (26) for TABLE RELATIVE
RETENTION MONO-
AND
TIMES
2 OF METHYLESTERS
DICARBOXYLIC
OF
ACIDS
Retention times, relative to hexadecanoic Acid” (n) = carbon numbers Hexanoic (6) Octanoic (8) Methylmalonic (4) Decanoic ( 10) Malonic (3) Fumaric (4) Succinic (4) Dodecanoic (12) Adipic (6) Tetradecanoic (14) Hexadecanoic (16) Octadecanoic (18) Octadecadienoic (18)
Monocarboxylic
Dicarboxylic
0.21 0.43 0.58 0.60 0.63 0.65 0.71 0.74 0.86 0.88 1.00 (19.93 min) 1.05 1.13
a Methyl esters of malic, ol-ketoglutaric. citric and isocitric acids were not determined due to insolubility in ether extracts prior to chromatography.
WARNER
AND VAHOUNY
FIG. 1. Gas-liquid chromatographic separation of methyl esters of mono- and dicarboxylic acids. Preparation of methyl ester derivatives and conditions for chromatography are described in the text.
isotope determination, the recovery of mass and radioactivity of methylmalonic, succinic and all long-chain acids was quantitative, as shown in Table 1. Table 2 summarizes the relative retention times of methyl esters of monocarboxylic acids and those dicarboxylic acids which were wholly or partially extracted and derivatized. It should again be emphasized that certain Krebs cycle intermediates, including malic, a-ketoglutaric, citric and isocitric acids, were partially or wholly lost during the procedure. However, in order to define the relative retention times of possible contaminants during quantitative determinations of methylmalonic and succinic acids, data on these acids have been included where possible. In addition, as shown in Fig. 1, the poor separation of malonic and fumaric acids on the chromatographic column also precludes use of this procedure for these particular intermediates. Figure 1 represents a composite gas-liquid chromatogram of methyl esters of mono- and dicarboxylic acids carried through the procedure and indicates that, whereas
METHYLMALONIC
AND
SUCCINIC
ACIDS
129
succinic acid is free of any contamination by other extracted acids, methylmalonic and decanoic acids have similar retention times. This monocarboxylic acid is not present in significant amounts in normal tissues, but during metabolic studies using radioactive substrates it becomes important to characterize further this chromatographic peak, preferably by mass spectrometry (e.g., see Ref. (5)). REFERENCES 1. Bamess, L. A., Moeksi, H., and Gyorgy, P. (1956)J. Viol. Chern. 221, 93. 2. Cox, E. V., and White, A. M. (1962) Lancer 2, 853. 3. Etlle, J. D., Clark, J. M., Jr.. Nystrom, R. F., and Johnson, B. C. (1964) J. Bid/. Chem. 239, 1920. 4. Giorgio. A. J., and Plaut, G. W. C. ( 1965) J. Lab. C/in. Med. 66, 667. 5. Sprinkle, T. J.. Porter, A. H., Greer, M.. and Williams, P. M. (1969) Clin. Chim. Acta 24, 416. 6. Smith, R. M., Osborne-White, W. S., and Russell, G. R. (1969) Biochem. J. 112, 703. 7. Bligh, E. G., and Dyer, W. J. (1959) Can. J. Eiochem. Physiol. 37, 911. 8. Ting, I. P., and Dugger, W. M., Jr. (1965) Anal. Biochem. 12, 571. 9. Kuksis, A., and Prioreschi. P. (1967) Anal. Biochem. 19, 468. 10. Bulen, W. A.. Vamer, J. E., and Burrell, R. C. (1952) Anal. Chem. 24, 187. 11. Morrison, M., and Stotz, E. (1955) .I. Biol. Chem. 213, 373. 12. Swim, H. E., and Utter. M. F. (1957) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. 0.. eds.), Vol. 4. p. 584. Academic Press, New York. 13. Denison, F. W., and Phares, E. F. (1952) Anal. Chem. 24, 1628. 14. Isherwood, F. A., and Hanes, C. S. (1953) Biochem. .I. 55, 824. 15. Cardinale, G. J., Dreyfus. P. M.. Auld, P., and Abeles, R. H. (1969) Arch. Biochem. Biophys. 131, 92. 16. Knappe, E., and Peteri, D. (1962) Z. Anal. Chem. 188, 184. 17. Stokke, O., Eldjain, L., Norum, K. R., Steen-Johnsen. J., and Halvorsen, S. (1967) Stand. J. C/in. Lab. Invest. 20, 313, 18. Barnett, D.. Cohen, R. D., Tassopoulos, C. N., Turtle. J. R., Dimitriadou. A.. and Fraser, T. R. (1968) Anal. Biochem. 26, 68. 19. Alcock, N. W. (1965) Anal. Biochem. 11, 335. 20. Rosenberg, L. E., Lilljeqvist, A., and Hsia. Y. E. (1968) Science 162, 805. 21. DuPont, J., and Mathias, M. M. (1968) Lipids 3, 545. 22. Travis, S., Mathias, M. M., and DuPont, J. (1971) Fed. Proc. 30, 520. 23. Vahouny, G. V., D’Amato. P. H., and Rodis, S. L. (1973) Lipids 8, 446. 24. Rodis, S. L., D’Amato, P. H., Koch, E., and Vahouny, G. V. (1970) Proc. Sot. Exp. Biol. Med. 133, 1070. 25. Beck, W. S., Flavin. M., and Ochoa, S. (1957) J. Biol. Chem. 229, 997. 26. Howard, C. F., Jr., and Kittinger, G. W. (1967) Lipids 2, 438. 27. Hyun, S. A., Vahouny, G. V., and Treadwell, C. R. (1965) Anal. Biochem. 10, 193.
BIOCHEMISTRY
67, I’?- 179 ( i 975)
Quantitative and Succinic
Extraction Acids and
by Gas-Liquid C. V. WARNER Department of Biochemistry,
of Methylmalonic Their Determination
Chromatography1 AND GEORGE
V. VAHOUNY
The George Washington University Medical Center, Washington, D.C. 20037
Received October 3, 1974: accepted February 10, 1975 A method for routine small-volume solvent extraction, derivitization and gasliquid chromatographic analysis of methylmalonic and succinic acids is described. The procedure allows for quantitative determination of mass and radioactivity of these acids and can be applied to metabolic studies of the genetic disorder methylmalonylacidurea.
Methods currently available for the extraction and determination of dicarboxylic acids, such as methylmalonic and succinic acids, are generally qualitative and cumbersome for routine application. Isolation of these acids from urine or tissue homogenates has often involved continuous, large-volume ether extractions (l-6) or chloroform and methanolwater extractions (7,8). Subsequent separation of these crude extracts is accomplished by ion-exchange or partition chromatography using large solvent-volumes (1,2,4,8-12), by paper chromatography (1,13,14,15), thin layer silicic acid chromatography (8,16,17), or, more recently, gasliquid chromatography (2,5,9,17- 19). With the exception of gas-liquid chromatography (glc), subsequent analysis of the acids following separation is by alkali titration (10) or, in the case of substituted malonic acids, by spectrophotometry following diazotization with p-nitroaniline (4). Accurate measurements of methylmalonic and succinic acids, as well as several other acids, have been hindered by the volatility of the unionized acids and their derivatives, which results in significant losses during concentration of the large volumes of solvent following extraction and during purification by chromatography. Cardinale et al. (1.5) described a spectrophotometric assay for methylmalonyl coenzyme A which is based on coupled reactions resulting in oxidation of NADH. However, these authors relied on less quantitative extraction and on paper chromatography for determination of isotopic methylmalonate. The problem of variable recoveries of organic acids can, in part, be corI This study was supported by Public Health Service Grant No. HL-09489.
Copyright @ 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
METHYLMALONIC
AND
SUCCINIC
ACIDS
123
rected by addition and recovery of radioactive carriers for each of the acids tested (18), but this precludes the measurement of metabolically derived acids from radioactive precursors. The report of methylmalonic acidurea as an inherited metabolic disorder (20) and the indication that biooxidation of polyunsaturated fatty acids may lead to formation of propionic acid and subsequently to methylmalonic and succinic acids (21,22) have emphasized the need for a more quantitative and relatively routine method for the extraction of these acids and for the quantitative analysis of mass and radioactivity of these acids during metabolic studies. The present report describes a technique for small-volume ether extraction of methylmalonic and succinic acids (as we11 as several other organic acids) which, when combined with gas-liquid radiochromatography following derivatization, permits routine and quantitative analysis of the specific radioactivities of these acids. EXPERIMENTAL
Materials The dicarboxylic acids were obtained from Calbiochem and fatty acids were from The Hormel Institute. [ 1,4-‘-C] Succinic acid was obtained from Amersham/Searle Corp. and [ 2-methyl-14C] malonic acid was purchased from New England Nuclear Corp. All solvents were distilled and reagents were of highest purity. Procedure 1. Extraction of organic acids. Since our studies have been concerned primarily with polyunsaturated fatty acids as potential sources of propionate, methylmalonate and succinate in heart and skeletal muscle, the procedure described here has been applied to in vitro incubation systems containing one or more radioactive compounds. These included linoleic, palmitic, propionic, methylmalonic and succinic acids, as well as certain other dicarboxylic acids described in the text. The incubation media for studies on biooxidation of fatty acids by whole tissue homogenates or mitochondria is described elsewhere (23). Incubations are carried out in capped 25-ml Erlenmeyer flasks containing a polyethylene center well. At the end of the incubation, hyamine hydroxide (0.3 ml) is injected through the rubber cap into the center well, and 0.23 ml of 6 N H&SO, is injected into the incubation medium. Incubation is continued at 37°C for 60 min to allow complete absorption of the evolved [‘“Cl 02, by hyamine, and the center well is removed and placed in 10 ml of scintillation cocktail in a liquid scintillation vial for determination of radioactivity (24).
124
WARNER
AND
VAHOUNY
The contents of the incubation vial are adjusted to pH 12 with NaOH to hydrolyze the CoA thioesters of the organic acids (25) and is then reacidified to pH 3.0 with H&SO+ Solid sodium chloride (ca. 400 mg) is added and the contents of the incubation flask are decanted quantitatively into a screw-cap tube using a total of 5 ml of diethyl ether. The biphasic mixture of ether and water is mixed thoroughly with a tube buzzer, the phases are allowed to separate, and the ether layer is transferred to a second screw-cap tube. The aqueous residue is extracted twice more in the same manner with 5-ml aliquots of ether, and the ether extracts are combined. When fatty acids are absent from the original incubation mixture, 1 mg of stearic acid is added to the ether extract to prevent volatilization losses of the short-chain dicarboxylic acids. Also, at this point, carrier (2 mg) methylmalonic and/or succinic acid is added when appropriate. Excess anhydrous Na,SO, is added to dry the solvent, the tubes are shaken and allowed to stand overnight. The ether extract is decanted into a third tube and the Na.SO, residue is washed twice with 3-5 ml of ether, combining the washings with the extract. The dehydrated extract is carefully evaporated to dryness in an ice bath under nitrogen; during the evaporation, the sides of the tube are frequently rinsed down with ether to reduce drying and evaporation of the acids. 2. Methylation. One milliliter of boron trifluoride-methanol reagent (14% by weight, Applied Science Co.) is added immediately after evaporation of the ether, the tubes are tightly capped and placed in a 70°C tube-heater for 2 hr. The tubes are cooled, 1 ml of distilled water is added, and the methyl esters are extracted with ether by the same procedure used above for extraction of the free un-ionized acids. This is done with three extractions using aliquots of 2 ml of ether twice and 1.5 ml of ether twice. The ether extracts are combined, dried with anhydrous sodium sulfate as before, and quantitatively transferred to calibrated centrifuge tubes. The ether is slowly evaporated to 1.0 ml in an ice-salt bath under nitrogen. Throughout the procedure, where possible, the tubes are maintained in ice baths to avoid volatilization of the methyl ester derivatives of the short-chain mono- and dicarboxylic acids. 3. Gas-liquid radioclzromatography. Gas chromatographic separation was carried out using a Beckman GC-55 gas chromatograph with a stream-splitter (1:20) and flame-ionization detectors. The glass column (6 ft X 0.25 in., o.d.) was packed with 15% HIEFF-2BP (ethylene glycol succinate) on IOO-200-mesh Gas-chrom P (Applied Science Co.). Analysis was carried out at helium carrier gas pressure of 20 psi, and injection block temperature was 250°C. Hydrogen flow was 45 cm3/min, and air flow was 270 cm3/min. Initial oven temperature was maintained at 60°C for 3 min, followed by a programmed temperature increase of
METHYLMALONIC
AND
SUCCINIC
ACIDS
125
7.5”C/min to 19o”C, which was maintained for 14 min for elution of long-chain polyunsaturated fatty acid derivatives. The horizontal arm from the stream-splitter for effluent gas collection passed through a 200°C block-heater (Barber-Coleman Co.) prior to trapping of the sample as described by Howard and Kittinger (26). The effluent gasses passed through a Pasteur pipet containing glass wool which had been treated with liquid scintillant ( 100 mg of 1,4-di-(2,5phenyloxazolyl)benzene and 4 gm of 2,5-diphenyloxazole per liter of toluene) and dried with nitrogen gas. The pipet was replaced at predetermined times during chromatography, based on simultaneous detection and recording of the chromatographic peaks. The contents of the pipet were then eluted into scintillation vials using a total of 10 ml of scintillation fluid. Radioactivity was determined on a three-channel Nuclear Chicago liquid scintillation spectrometer (Mark I). RESULTS
AND
DISCUSSION
Gas chromatography has been successfully applied to the detection of short- and long-chain monocarboxylic acids (e.g., 2,5,9,17-19,27). However, before glc can be applied to biological materials, some degree of purification of the materials is necessary (12). Due to the variable volatility of these shorter-chain acids and their low concentrations in biological materials, the solvent extraction and evaporation procedures generally result in significant losses even before analysis can be achieved (9,18). These losses are further exaggerated prior to gas chromatography after derivatization of the acids to the less polar methyl esters (9). For example, it has been reported that up to 90% of methyl lactate and 70% of trimethyl citrate may be lost by evaporation of a chloroform extract (18). Several techniques have been reported to overcome some of these problems. Stokke et al. (17) and Cardinale et al. (15) have described methods for extraction of methylmalonate from urine or tissue homogenates using small volumes of diethyl ether (9-l 6 ml); reduced losses of volatile acids during evaporation of solvents or during column chromatography have been accomplished by addition of methyl pelargonate. which can also serve as an external standard for glc (2). As mentioned earlier, the addition of radioactive standards to correct for losses throughout extraction and purification procedures has been suggested by Barnett et al. (IS), and Sprinkle et al. (5) have employed the ditrimethylsilyl ester of methylmalonate for glc in order to avoid ionexchange or thin layer chromatography for preliminary purification of ether extracts of urine. The procedure presented in this communication includes: (a) The use of small volumes of diethyl ether for quantitative extraction of methylmalonic and succinic acids from biological materials; (b) the addition of
126
WARNER
AND VAHOUNY
octadecanoic acid to reduce the volatility of the short-chain dicarboxylic acids and their methyl esters during the procedure; (c) the controlled evaporation of diethyl ether solvent containing methyl esters of these acids; and (d) the use of gas-liquid chromatography for quantitative determination and radioactivity of these compounds. This procedure has proven to be a useful routine method for the separate measurements of mass and radioactivity employed previously. The data in Table 1 summarize the recoveries of added labeled methylmalonic and succinic acids from mitochondrial incubation media or tissue homogenates. It is apparent that the undissociated acids (pH 2.0) are quantitatively extracted into small volumes (20 ml) of diethyl ether without the necessity for continuous extraction procedures. For metabolic studies, the procedure initially allows quantitative recovery of [‘“Cl 0, by acidification of the mitochondrial incubation medium to pH 2 and, following adjustment of the medium to pH 12 to hydrolyze the coenzyme A thioester bonds and extraction of the medium at pH 3 with ether, provides quantitative recovery of certain metabolic intermediates. When the incubation medium contained long-chain fatty acids or if carrier octadecanoic acid was added prior to extraction, there were no losses of methylmalonic and succinic acids during evaporation of the iniTABLE RECOVERY
OF METHYLMALONIC DERIVATIZATION
1
AND SUCCINIC AND GAS-LIQUID
ACIDS DURING CHROMATOGRAPHY
EXTRACTIONS,
% Recovery of cL Procedures 1. Ether extraction, pH 2-3 incubation medium 2. Extraction and evaporation of ether extracts a. In absence of carrier octadecanoic acid b. In presence of carrier octadecanoic acid 3. Derivatization, ester extraction, and evaporation of ether 4. Complete procedure (steps 1, 2b. 3) 5. Gas-liquid tography
A Methylmalonic
B Succinic acid
A and B mixture
98 2 1
101 k 1
96 2 5
90 f 1
91 2 1
93 2 3
97
99
91 i 1
88 2 1
91 F 2
93 * 1
85 r 2
88 f 1
100.7 k 2.7
97.9 f 2.0
104
acid
radiochroma-
a The values represent means 2 SEM for up to ten determination.
-
METHYLMALONIC
AND
SUCCINIC
127
ACIDS
tial ether extracts under nitrogen in preparation for derivatization (Table 1, Procedure 2b). However, in the absence of carrier, significant volatilization of these acids occurred, especially during the final stages of evaporation. This could, in part, be prevented by maintaining the extracts in ice during the entire procedure. Derivatization with BF,-methanol and subsequent extraction of methyl esters have been effectively employed earlier for short- and longchain fatty acids and presented no unique problems with methylmalonic and succinic acids (Table 1, Procedure 3). However, evaporation of the ether extracts of the dimethyl esters of these acids was the major source of losses in the entire procedure. These evaporations were carried out in ice, but it was found that significant losses occurred during complete evaporation of the solvent. By carefully and regularly washing down the sides of the tube with small volumes of ether, and preventing the volume of solvent to fall below 1.0 ml, it was possible to avoid major losses of the two short-chain dicarboxylic acids (Table 1, Procedure 4). This final evaporation step is then the major limitation in the procedure in terms of providing a concentrated preparation of derivatized methylmalonic and succinic acids for gas-liquid chromatography. The same limitation is expected with the trimethylsilyl derivatives (5) of these acids. Using a 20: 1 stream-splitter, in which the major column effluent was trapped (26) for TABLE RELATIVE
RETENTION MONO-
AND
TIMES
2 OF METHYLESTERS
DICARBOXYLIC
OF
ACIDS
Retention times, relative to hexadecanoic Acid” (n) = carbon numbers Hexanoic (6) Octanoic (8) Methylmalonic (4) Decanoic ( 10) Malonic (3) Fumaric (4) Succinic (4) Dodecanoic (12) Adipic (6) Tetradecanoic (14) Hexadecanoic (16) Octadecanoic (18) Octadecadienoic (18)
Monocarboxylic
Dicarboxylic
0.21 0.43 0.58 0.60 0.63 0.65 0.71 0.74 0.86 0.88 1.00 (19.93 min) 1.05 1.13
a Methyl esters of malic, ol-ketoglutaric. citric and isocitric acids were not determined due to insolubility in ether extracts prior to chromatography.
WARNER
AND VAHOUNY
FIG. 1. Gas-liquid chromatographic separation of methyl esters of mono- and dicarboxylic acids. Preparation of methyl ester derivatives and conditions for chromatography are described in the text.
isotope determination, the recovery of mass and radioactivity of methylmalonic, succinic and all long-chain acids was quantitative, as shown in Table 1. Table 2 summarizes the relative retention times of methyl esters of monocarboxylic acids and those dicarboxylic acids which were wholly or partially extracted and derivatized. It should again be emphasized that certain Krebs cycle intermediates, including malic, a-ketoglutaric, citric and isocitric acids, were partially or wholly lost during the procedure. However, in order to define the relative retention times of possible contaminants during quantitative determinations of methylmalonic and succinic acids, data on these acids have been included where possible. In addition, as shown in Fig. 1, the poor separation of malonic and fumaric acids on the chromatographic column also precludes use of this procedure for these particular intermediates. Figure 1 represents a composite gas-liquid chromatogram of methyl esters of mono- and dicarboxylic acids carried through the procedure and indicates that, whereas
METHYLMALONIC
AND
SUCCINIC
ACIDS
129
succinic acid is free of any contamination by other extracted acids, methylmalonic and decanoic acids have similar retention times. This monocarboxylic acid is not present in significant amounts in normal tissues, but during metabolic studies using radioactive substrates it becomes important to characterize further this chromatographic peak, preferably by mass spectrometry (e.g., see Ref. (5)). REFERENCES 1. Bamess, L. A., Moeksi, H., and Gyorgy, P. (1956)J. Viol. Chern. 221, 93. 2. Cox, E. V., and White, A. M. (1962) Lancer 2, 853. 3. Etlle, J. D., Clark, J. M., Jr.. Nystrom, R. F., and Johnson, B. C. (1964) J. Bid/. Chem. 239, 1920. 4. Giorgio. A. J., and Plaut, G. W. C. ( 1965) J. Lab. C/in. Med. 66, 667. 5. Sprinkle, T. J.. Porter, A. H., Greer, M.. and Williams, P. M. (1969) Clin. Chim. Acta 24, 416. 6. Smith, R. M., Osborne-White, W. S., and Russell, G. R. (1969) Biochem. J. 112, 703. 7. Bligh, E. G., and Dyer, W. J. (1959) Can. J. Eiochem. Physiol. 37, 911. 8. Ting, I. P., and Dugger, W. M., Jr. (1965) Anal. Biochem. 12, 571. 9. Kuksis, A., and Prioreschi. P. (1967) Anal. Biochem. 19, 468. 10. Bulen, W. A.. Vamer, J. E., and Burrell, R. C. (1952) Anal. Chem. 24, 187. 11. Morrison, M., and Stotz, E. (1955) .I. Biol. Chem. 213, 373. 12. Swim, H. E., and Utter. M. F. (1957) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. 0.. eds.), Vol. 4. p. 584. Academic Press, New York. 13. Denison, F. W., and Phares, E. F. (1952) Anal. Chem. 24, 1628. 14. Isherwood, F. A., and Hanes, C. S. (1953) Biochem. .I. 55, 824. 15. Cardinale, G. J., Dreyfus. P. M.. Auld, P., and Abeles, R. H. (1969) Arch. Biochem. Biophys. 131, 92. 16. Knappe, E., and Peteri, D. (1962) Z. Anal. Chem. 188, 184. 17. Stokke, O., Eldjain, L., Norum, K. R., Steen-Johnsen. J., and Halvorsen, S. (1967) Stand. J. C/in. Lab. Invest. 20, 313, 18. Barnett, D.. Cohen, R. D., Tassopoulos, C. N., Turtle. J. R., Dimitriadou. A.. and Fraser, T. R. (1968) Anal. Biochem. 26, 68. 19. Alcock, N. W. (1965) Anal. Biochem. 11, 335. 20. Rosenberg, L. E., Lilljeqvist, A., and Hsia. Y. E. (1968) Science 162, 805. 21. DuPont, J., and Mathias, M. M. (1968) Lipids 3, 545. 22. Travis, S., Mathias, M. M., and DuPont, J. (1971) Fed. Proc. 30, 520. 23. Vahouny, G. V., D’Amato. P. H., and Rodis, S. L. (1973) Lipids 8, 446. 24. Rodis, S. L., D’Amato, P. H., Koch, E., and Vahouny, G. V. (1970) Proc. Sot. Exp. Biol. Med. 133, 1070. 25. Beck, W. S., Flavin. M., and Ochoa, S. (1957) J. Biol. Chem. 229, 997. 26. Howard, C. F., Jr., and Kittinger, G. W. (1967) Lipids 2, 438. 27. Hyun, S. A., Vahouny, G. V., and Treadwell, C. R. (1965) Anal. Biochem. 10, 193.
