Analysis of Krebs Cycle and Related Acids in Guinea Pig Tissues by Gas-Liquid Chromatography Esther Mensen de Silva The Department of Physiological Sciences, Section of Biochemistry, Unioersidad Peruanu Cayetano Heredia, Lima, Peru Acids of the Krebs cycle were determined in the heart and skeletal muscle and liver and kidney of uinea pigs by gas-liquid ch romatog raphy (GLC). Pissue samples weighing 0.5 to 2.0 rams were first extracted with a methanol: water: H2$, solution. Purification of the extract was made by eluting the or anic acids with ether from a 25-gram column made o! a mixture of Celite, anhydrous Na2S04, and the extract. The organic acids were collected on a 1-gram column of basic alumina placed below the Celite column, and the acids were methylated by treating the alumina with BF3-methanol. Fatty-acid methyl esters were separated as a group from the Krebs-cycle methyl esters by extraction into heptane. The methyl esters were completely separated in 14 min using temperature programming on a 3-ft x 1-mm (i.d.) column packed with 5% polyethylene glycol adipate on silanized Celite. The response of the hydrogen flame ionization detector was linear for the 1-MIsamples injected containing 0.1 to 2.5 pg of each methyl ester. Mean recoveries of standards from distilled water and tissues were similar, but varied for each individual acid from 40.9 to 96.0%. The relative standard deviation for three replicate analyses ranged from 2.7 to 24.5%.
the use of such columns requires a long time period for the elution of all the methyl esters, unless excessively high column temperatures are applied. This paper describes an effective method for extraction, purification, and analysis by gas chromatography of the intermediates present in 0.5- to 2.0-gram samples of liver, kidney, heart, and skeletal muscle, with fair and reproducible recoveries. The method uses a modification of the Celite column described by Swim and Utter (13), combined with a small column of activated basic alumina on which the acids are collected and directly methylated with BFamethanol. Separation of the fatty acids from the Krebscycle acids, by heptane extraction, is done following their methylation, but prior to G L C analysis. Complete GLC separation of the methyl esters of lactic, pyruvic, malonic, fumaric, succinic, adipic, malic, alpha-keto-glutaric, cisaconitic, citric and isocitric standard acids is obtained in 14 min on a column 3 ft in length with temperature programming from 80-180 O C . The methyl esters of malonic and adipic acids are used as internal standards.
PREVIOUSLY reported methods (1, 2) for the isolation of Krebs-cycle acids from animal tissues for gas chromatography, gave poor and variable recoveries when estimations on small amounts of tissue were attempted. Kuksis and Prioreschi (1) were the first to describe the preliminary separation and purification of the natural acids from animal tissues by means of thin-layer chromatography, among other methods, showing excellent recoveries in amounts of tissue not less than 5 grams. Barnett et al. ( 2 ) have described a simple method for isolation of these acids from smaller amounts of tissue (0.25 gram), but with great losses. They were able to correct for the low and variable recovery by using a radioactive standard for each acid measured. In order to obtain the complete separation by gas chromatography of a mixture of the organic acid methyl esters, previous authors (1-12) have reported the necessity of using packed columns at least 5 o r 6 feet in length. However,
EXPERIMENTAL
(1) A. Kuksis and P. Prioreschi, Aiial. Biochem., 19,468 (1967). (2) D. Barnett, R. D. Cohen, C. N. Tassopoulos, J. R. Turtle, A. Dimitriadou, and T. R. Fraser, ibid., 26, 68 (1968). (3) A. Kuksis and P. Vishwakarma, Cali. J . Biochem. Physiol., 41, 2353 (1963). 35, (4) H. H. Luke, T. E. Freeman, and L. B. Kier, ANAL.CHEM., 1916 (1963).
(5) H. M. Kellogg, E. Brochmann-Hanssen, and A. B. Svendsen J . Pliarm. Sei., 53, 420 (1964). (6) T. S.Rumsey, C. H. Noller, J. C. Burns, D. Kalb, C. L. Rhykerd, and D. L. Hill, J. Dairy Sci., 47, 1418 (1964). (7) D. W. Alcock, Alia/. Biochem.. 11. 335 (1965). (8) Z. Horii, M. Makita, and Y. Tamura, Chem. hid. (Loiido/i),34, 1494 ( 1965). (9) D. T. Canvin, Cau. J . Biochem., 43, 1281 (1965). (10) F. L. Estes and R. C. Bachrnann, ANAL. CHEM.,38, 1178 (1966). (11) C. E. Dalgliesch, E. C. Homing, M. G. Homing, K. L. Knox, and K . Yarger, Biochem. J . , 101,792 (1966). (12) D. S.Zaura and J. Metcoff, ANAL.CHEM., 41,1781 (1969).
Reagents. Standard organic acids of the highest available purity, including the TCA cycle acids, related organic acids, fatty acids, and fatty-acid methyl esters were obtained from Sigma Chemical Co. (U. S. A.). Lactic acid was in the crystalline form and isocitric as the trisodium salt. Other reagents were: 51 BF8-methanol (British Drug Houses, Great Britain); Celite 545 (Johns-Manville, U.S.A.); Celite (Gas-Pack W-untreated) 100-120 mesh (Chemical Research Service, U. S. A.); dimethyldichlorosilane (DMCS) (Applied Science Laboratories, U. S. A.). The Celite 545 was washed with ether as described by Swim and Utter (13). A 8 % solution of BF3 in methanol was made by diluting the 51 % solution with dry methanol, which was prepared as described by Vogel (14). All organic solvents were analytical grade and found to be suitable for GLC. Procedure. MATERIALSFOR GAS CHROMATOGRAPHY. Preparation of the Celite support was done according to the procedure described by Link et at. (13, and polyethylene glycol adipate (PEGA) was made using the method of Farquhar et al. (16). The granules of Celite were silanized by exposure t o DMCS vapors in a closed container for 2 weeks, then washed with methanol, and coated with PEGA ( 5 % wlw). The animal tissues were obtained from male guinea pigs native to sea level, maintained under controlled conditions of diet and temperature for 3 months and weighing 500-600 grams. Light anaesthesia was produced by the intraperi(13) H. E. Swim and M. F. Utter, in “Methods in Enzymology,” Vol. 4, S. P. Colowick and N. 0. Kaplan, Academic Press, New York, 1957, p 584. (14) A. I. Vogel, “Practical Organic Chemistry,” 3rd ed., Longmans, London, 1962, p 167. (15) W. E. Link, R. A. Morrissette, A. D. Cooper, and C. F. Smullin, J. Amer. Oil Chem. SOC.,37, 364 (1960). (16) J. W. Farquhar, W. Insull, Jr., P. Rosen, W. Stoffel, and E. H. Ahrens, Jr., Nufrifioii Reu., 17, Suppl. 1-30 (1959). ANALYTICAL CHEMISTRY, VOL. 43, NO. 8, JULY 1971
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Figure 1. Gas-liquid chromatography of methyl esters of Krebs-cycle acids and fatty acids, utilizing a glass column 3 f t X 1 m m (i.d.) containing 5 PEGA on silanized Gas Pack W (100-120 mesh) previously washed with acid, base, alcohol, and chloroform. Carrier gas flow 40 ml/min, constant temperature at 80°Cfor 1.33 min, following by temperature programming to 180 "C at rate of 10"/min (A) Reference standard mixture containing approximately 10 mg total acids. (B) Guinea-pig kidney extract of Krebs-cycle acids from 2 grams of tissue. (C) Guinea-pig kidney extract of fatty-acid methyl esters found in the first heptane wash corresponding to tissue sample shown in chromatograph (B). Peaks identified as follows are the peaks obtained from methylating the corresponding acids: 1)lactic acid, 2) pyruvic acid, 3) internal standard malonic acid, 4) fumaric acid, 5) succinic acid, 6) internal standard adipic acid, 7) malic acid, 8) and 9) alpha-keto-glutaric acid, 10) cis-aconitic acid, 11)citric acid, 12) isocitric acid, 13) unidentified peak, a) lauric acid, b) myristic acid, c ) palmitic acid, d ) palmitoleic acid, e) stearic acid,f)oleic acid, g)linoleic acid toneal injection of sodium pentabarbital (2.5 mg/100 grams), followed by 1.32 units curare/100 grams (E. R. Squibb & Sons) to prevent muscular contractions. When respiratory paralysis set in, by 3 to 5 min, death was produced by breaking the neck. Tissues were removed 10 min after death, the purpose being to obtain samples under conditions of severe anoxia. The tissue was clamped with flat tongs, previously cooled in liquid nitrogen, and the tissue was stored and pulverized in liquid nitrogen. The grinding and weighing of all tissues was done in a cold box at -50" t o -60 "C. The cold box was a modification by Krumdieck (17) of Peterson's apparatus (18). EXTRACTION OF ACIDS. Frozen pulverized samples (0.5 to 2.0 grams) of guinea-pig heart and skeletal muscle, kidney and liver were homogenized in 10 sec with 10 ml of cold
(17) A. Cazorla and C. Krumdieck, in "Cellular and Tisular Adaption to Hypoxia," Report No. 1, Army Project No: 2N014501B71D 00 031 LA, U.S. Army Element (1967). (18) R. D. Peterson. D. Gaudin. P. M. Bocek, and c. H. Beattys, Amer. J. Physiol.,'206, 599 (1964). 1032
ANALYTICAL CHEMISTRY, VOL. 43, NO. 8, JULY 1971
(-70 "C) 5N H2S04:water:methanol (1:3:16 v/v/v) using a Sorvall OmniMixer (Ivan Sorvall, Norwalk, Conn.) while cooling the tubes continuously with dry ice. The homogenate was centrifuged at 0", and the residue was homogenized once more with 10 ml of 5N HzS04:water:methanol. With the aid of a stream of nitrogen, the supernatant was reduced to a volume equivalent to the approximated volume of total water in the supernatant, and saved for subsequent purification. Mixtures of standard acids were dissolved in 20 ml of 5N H2S04:water:methanol and 2 ml of water. The solution was evaporated down to remove the methanol and saved for further treatment. The range tested was approximately 2-20 mg total for 9 acids. The individual amounts of acids ranged from 0.05-6.0 mg. In addition, mixtures of standard acids (2-20 mg total) were dissolved in 20 ml of 5N H 8 0 4 :water : methanol and this 20 ml was used to homogenize 2 grams of tissue' PURIFICATION OF TISSUE EXTRACTS.The following procedure for purification of the crude methanol-acid extract was developed in order to remove all interfering substances and reduce the background in the gas chromatographs t o
Table I. Recovery of Standard Acid Mixtures from Extraction and Purification Procedure A B Recovery of standard mixture of maximal) Detector response (peak area) cm2/pg methyl ester From distilled water From tissue homogenate Re1 std dev Mean" Re1 std dev Meana Re1 std dev Meano
(z
Acid Lactic 24 11.0 68.5 12.1 12.1 13.9 Pyruvic 155 4.0 5.3 24.5 1.6 14.4 Malonic 56 0.7 Fumaric 101 0.5 56.3 6.8 55.0 1.6 Succinic 100 0.3 96.0 2.1 94.9 5.3 Adipic 100 0.3 Malic 48 0.8 68.7 7.1 68.0 11.6 alpha-k-Glutaric 101 0.5 45.3 18.1 40.9 22.3 cis-Aconit ic 112 0.6 43.4 7.9 43.1 14.1 Citric 100 0.9 82.5 8.1 82.6 6.4 Isocitric 40 0.6 58.5 13.0 58.1 10.5 a Mean of 3 determinations. A. Dry standard acid mixtures (90-190 mg total) were methylated with 8 % BF8-methanol, and the methyl esters formed were extracted directly into chloroform. A 1-pl aliquot of 10 ml total was injected onto the chromatographic column. Values represent the best methylation achieved under the conditions used, and recovery of the esters formed. B. Standard acid mixtures (2-20 mg total) were taken through the extraction and purification procedure. The recovery was calculated by comparing the peak areas found with the data shown in Column A. Internal standards of dimethyl malonate and dimethyl adipate were used to correct for final volume variation (10-50 pl) of the sample. Partial loss of fumaric and cis-aconitic was due to their extraction into heptane.
the level obtained with the pure standard solutions. The crude acid extract was mixed thoroughly with ether-washed Celite 545 (2 grams of Celite/ml of extract). The moist Celite was then mixed with anhydrous Na2S04(2 grams/ml of extract) and stored overnight under vacuum in a desiccator containing P20s. The powder mixture was transferred to a 50 cm X 1 cm ( i d . ) glass chromatographic column in approximately 2-gram portions and packed down tightly. Because of the addition of anhydrous Na2S04,the organic acids were eluted from the column in the .first 100 ml of ether instead of 450-500 ml as reported by Swim and Utter (IS). A pressure of 15 psi was necessary t o maintain a flow of 1 ml/min. A 1-gram column of activated basic aluminum oxide was placed at the bottom of the Celite column to trap the organic acid fraction as the ether eluate passed through it. G L C analysis of the ether eluate of tissue extracts showed chromatograms with high backgrounds and very high broad peaks, demonstrating the elimination a t this point, of most of the remaining material which interferes with the G L C determination of the Krebs-cycle acids. Thorough drying of the alumina, by removing the small amount of water that passed off the Celite column, increased 2.5- to 4.5-fold the degree of methylation of the acids, and was accomplished by repeated washings with acetone (five) and ether (two). Conversion of the acids into their methyl esters was achieved by heating the BFa-methanol to 60 "C for 20 min. alumina with 5 ml of Following methylation, the mixture was cooled to room temperature and filtered, and a n equal volume of water was added t o the filtrate. This filtrate contained methyl esters of fatty acids and Krebs-cycle acids, and the fatty-acid methyl esters were removed by washing the methanol-BF,water solution with 1 ml of n-heptane, 3 times. The Krebscycle methyl esters were extracted with two 10-ml portions of chloroform, and the chloroform extract was washed with a 1M N a 2 C 0 3solution, followed by water until neutral, and dried over anhydrous Na2S04. A known amount of dimethyl malonate and/or dimethyl adipate (internal standard) was added and the chloroform extract was reduced to a 10-50 pl volume. A 1-111 aliquot was injected into the gas-liquid chromatograph column. GASCHROMATOGRAPHY. The first instrument used was a Research Specialist Argon Chromatogram fitted with a betaray ionization detector. The second instrument was a PerkinElmer 881 temperature-programmed gas chromatogram with
8z
a dual flame ionization detector. Both instruments were used with a Honeywell -0.2 to 1.0 mV recorder. G L C determination of the methyl esters was carried out as described under Figure l . Quantitation. Throughout the entire range of 0.01 pg t o 10.0 pg, a linear relationship passing through the origin was observed between the detector response and the quantities of individual standard esters injected. Over the attenuation ranges covered for G L C analysis of the tissues and standard acid mixtures the I-pl volumes contained 0.1 to 2.5 Hg of each methyl ester. The final chloroform extraction of tissue samples with or without the addition of known amounts of organic acids, or water with known amounts of organic acids varied in volume from 10-50 pl. This volume variation was corrected for by adding a known amount of internal standard before concentrating the sample. The peak areas were then compared with the peak areas of both individual known pure acids and their standard mixtures, methylated directly with BFsmethanol and extracted into chloroform. Using this direct comparison, the recovery from the extraction and purification procedure was calculated and the tissue concentration levels were estimated. RESULTS AND DISCUSSION
Recovery of Standard Acids from Tissues. Homogeniza. tion of the tissue in a mixture of methanol, water, and acid showed a marked increase in the recovery of the Krebs-cycle acids and a lower concentration of interfering substances, when compared with previous homogenization methods using ether, acetone, chloroform, methanol, or combinations of these solvents. The use of alumina to collect the acids avoids loss of the more volatile acids, by eliminating the necessity of evaporating a solution to dryness, in order to methylate the acids quantitatively. Figure l(C), a chromatogram of the first heptane extract of fatty. acid esters separated during the final purification step, and identified by comparing their retention times against pure standards, shows several peaks with similar retention times as the Krebs-cycle intermediates. The main interference is due to linoleic acid (peak g), which is not separated from citric acid (peak l l ) , and myristic acid (peak b), which is separated ANALYTICAL CHEMISTRY, VOL. 43, NO. 8, JULY 1971
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Table 11. Average Concentration of Krebs-Cycle Acids in Guinea Pigs Native to Sea Level Following 10 Minutes of Acute Anoxia &gram wet weight (mean =tS.E.M.)" Acid Heart Muscle Liver Fumaricb 1.7 1.3 1.1 Succinic 5.8 + 1 . 5 1 5 . 1 =t 2 . 5 4.2 f 1.4 Malic 67.3 f 9 . 4 49.3 f 9 . 0 49.6 i 15.7 Alpha-keto-glutaricb 1.4 0.7 1.2 cis-Aconiticb
