- Email: [email protected]
[13] Measurement of carnitine and O-acylcarnitines
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Conclusions Many important studies have been conducted with the reductive ozonolysis of fatty acids. Among the different reductive reagents, triphenylphosphine is the most widely used. During reductive ozonolysis, aldehydes, dialdehydes, and aldehyde esters are formed. The aldehyde esters and dialdehydes are less stable than the corresponding acid methyl esters and are usually not commercially available as reference materials. Therefore, these components may need to be synthesized prior to the GLC analyses. The different reductive techniques have undergone a variety of modifications and improvements, but some problems still remain with the GLC analysis of the aldehydic fragments due to chemical residues and also, apparently, problems of overlap between aldehyde and aldehyde esters. Fewer examples of application are as yet available for high-yield oxidative ozonolysis. The method described by the authors is simple and gives excellent yields of the principal acidic products. There is no reason not to apply oxidative ozonolysis in BF3-MeOH to sterols, hydrocarbons, and similar compounds. This method of ozonolysis of methyl esters in BF3-MeOH can be applied directly to monoethylenic and methylene-interrupted di-, tri-, and tetraethylenic fatty acids and apparently also to acetylenic bonds of fatty acids. In the case of non-methyleneinterrupted fatty acids, it became necessary to convert the methyl ester into an alcohol prior to the ozonolysis. When non-methylene-interrupted di-, tri-, and tetraethylenic acids needed to be identified, the authors effected the partial hydrazine reduction of these trienes and tretaenes, isolated the resulting monoethylenic acids, and did the ozonolysis on these simpler fatty acids. It should also be noted that this multiple-operation analysis can give the geometric configuration of particular ethylenic bonds, information not otherwise available for most polyethylenic fatty acids. The combination of BF3-MeOH oxidative ozonolysis and opentubular GLC chromatography on SILAR-5CP, SILAR-7CP, or BDS, which gives an excellent separation of the mono- and diesters, is a powerful tool for structural determination of unsaturated fatty acids.
[13] Measurement of C a r n i t i n e a n d O - A c y l c a r n i t i n e s By L. L. BIEBER and L. M. LEWlN A half century after its isolation from meat, t carnitine was shown to be essential for fl-oxidation of long-chain fatty acids in mammalian sysW. G u l e w i t s c h a n d R. K r i m b e r g ,
METHODS IN ENZYMOLOGY, VOL. 72
Hoppe-Seyler's Z. Physiol. Chem.
45, 326 (1905).
Copyright © 1981 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181972-8
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tems. z,a More recently, the recognition that some human muscle myopathies may be induced by carnitine deficiencies or deficiencies in carnitine acyltransferase activity, and the recognition that carnitine may have more than a single role in intermediary metabolism 4-6 has led to the refinement and development of methods for the measurement of total carnitine and its O-acyl derivatives. This chapter describes some of the newer methodology and some modifications of the older methods but does not repeat the methods described in a previous volume, r
Assay for Free Carnitine, Total Carnitine, and Water-Soluble Acylcarnitines Enzymatic Method Principles
The assays for carnitine are based on the reaction l-Carnitine + acetyl-CoA ~
acetyl-l-carnitine + C o A S H
catalyzed by carnitine acetyltransferase (EC 2.3.1.7). 7 The sensitivity o f the enzymatic method was greatly increased by the introduction of a radioisotopic assay 8 that depends on the incorporation of the acetyl moiety of [1-14C]acetyl-CoA into acetylcarnitine followed by separation and measurement of the amount of [14C]acetylcarnitine formed. The method can be used for free carnitine, total carnitine, and O-acylcarnitines after hydrolysis. The difference between total carnitine and free carnitine yields the amount of O-acylcarnitines. Procedure
Perchloric acid extracts o f weighed tissue are made as described in a previous volume 7 or as described in this chapter in the section on quantitation of acylcarnitines. The extract is divided equally or aliquots are taken; one is neutralized with K O H to precipitate KCIO4, and the others are made 1.0 N with 2 N KOH. The alkaline samples are warmed to 40° for 30 2 I. B. Fritz, Adv. Lipid Res. 1,285 (1963). 3 j. Bremer, J. Biol. Chem. 237, 3628 (1962). 4 M.. E. Mitchell, Am. J. Clin. Nutr. 31, 645 (1978). Y. R. Choi, P. R. H. Clarke, and L. L. Bieber, J. Biol. Chem. 254, 5580 (1979). 6 D. J. Pearson and P. K. Tubbs, Biochem. J. 105, 1953 (1967). r D. J. Pearson, J. F. Chase, and P. K. Tubbs, this series, Vol. 14, p. 612. 8 G. Cederblad and S. Lindstedt, Clin. Chim. Acta 37, 235 (1972).
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min to saponify the short-chain, water-soluble acylcarnitines and then are neutralized with HCIO4. All neutralized samples are chilled for 30 min on ice and then centrifuged to r e m o v e KC104. Aliquots o f the neutralized supernatant fluids are then assayed for carnitine. The protein residue from the perchloric acid extract is suspended in 2 N K O H for 2 hr at 80° and the mixture is then neutralized with HCIO4 and processed as described above. This represents the long-chain acylcarnitine fraction. A modification o f the original C e d e r b l a d - L i n d s t e d t 8 assay is described below. The following stock solutions are made: A: Tris-DTNB containing 0.4 M Tris-HC1, 20 m M D T N B ; p H 7.3 B: Carnitine acetyltransferase; the commercial e n z y m e is diluted 1 : 10 prior to use (0.5 mg/ml) C: [14C]Acetyl-CoA, 3 . 4 5 / z M , 0.2/zCi/ml D: Acetyl-CoA, 0.1 m M Solutions A, C, and D are stored frozen at - 2 0 ° and solution B is made daily from commercial e n z y m e preparations. Prior to assay, two volumes o f solution C are mixed with one volume o f solution A and one volume o f stock solution D to make a total volume sufficient for 100 p.l per assay. After addition o f 100/zl o f assay mixture to 6 x 58 mm glass tubes, then 0, 0.4, 0.8, 1.2, 1.6, and 2.0 nmol of standard l-carnitine (0.10 m M 1-carnitine; 10 /zl/nmol) are added to individual tubes or aliquots o f solutions to be assayed, and distilled water is added to yield a total volume o f 200 p.l. The reaction is started by adding 20 p.l o f e n z y m e solution to make a final volume of 220/.d. Incubations are carried out at 37 ° for 30 min. Next, 200/zl from each tube are pipetted onto a 5 x 35 mm D o w e x 1, C1- column ( 8 x , 200-400 mesh), and the effluent is collected in a scintillation vial. The column is washed twice with 0.5 ml o f distilled water. The entire effluent is counted, and the amount o f carnitine per sample is calculated using the experimentally determined standard curve. Total carnitine = water-soluble (free carnitine) + short-chain (water-soluble acylcarnitine fraction) + long-chain acylcarnitine fraction. Remarks This method provides a measurement o f the total amount of shortchain acylcarnitines, but does not identify the acyl groups. Measurement o f individual acylcarnitines requires separation, identification, and quantitation of the individual components of an acylcarnitine mixture. Methods for both separation and quantitation of the short-chain acylcarnitines are described below. The C e d e r b l a d - L i n d s t e d t assay s has been modified because the presence o f low-molecular-weight short-chain acylcarnitines, such as acetylcarnitine, causes nonlinear standard curves and can cause an overestima-
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tion of the amount of free carnitine in relation to total carnitine by exchange of [14C]acetyl residues from [14C]acetyl-CoA into the acylcarnitine pool. This limitation can be overcome either by increasing the ratio of acetyl-CoA to carnitine 9'~° in order to force the reaction to the right or by adding a reagent to trap free CoA, which pulls the reaction to the right. D T N B 2 sodium tetrathionite H and N-ethylmaleimide ~2 have all been used by different investigators as trapping agents. The method described here uses DTNB, and one of us (L. Lewin) has used the N-ethymalemide modification with good success. Neither of us has used tetrathionate; consequently we do not recommend one method over the other. N-Ethylmaleimide ~2 and tetrathionate H apparently do not inactivate carnitine acetyltransferase as rapidly as DTNB. Microbiological Assay for Total 1-Carnitine
Principle The growth rate of a carnitine-requiring mutant of the yeast Torulopsis bovina can be dependent on the carnitine content of the growth medium when cultures are grown under strictly defined conditions.
Reagents and Organism Assay Organism. A carnitine-requiring strain of the yeast Torupopsis bovina can be obtained from the American Type Culture Collection, Rockville, Maryland (ATTC No. 26014) or from the Centraalbureau voor Schimmelkultures, Delft, Holland (CBS No. 6471). Basal medium. The basal medium is composed of solutions A and B. To prepare double-strength basal medium, solution A (250 ml) and solution B (10 ml) are mixed and brought to a final volume of 500 ml. Solution A: This contains (in g/liter): glucose, 80; L-asparagine - H20, 4.0; KH2PO4, 2.0; MgSO4 7 H20, 2.0; NaC1, 0.4; adenine, 0.04; cytosine, 0.04; oL-tryptophan, 0.20; L-phenylalanine, 0.32; and De-methionine, 0.08. To facilitate solution, the adenine, cytosine, and amino acids are dissolved in a small amount of 1N HCI. Potassium phosphate and magnesium sulfate are dissolved separately and care is taken in mixing the final solution to prevent precipitation of magnesium phosphate. This solution is adjusted to pH 5.0 and stored at 4° under toluene. T. B 0 h m e r , A. Rydning, a n d H. E. Solberg, Clin. Chim. Acta 57, 55 (1974). ~0 j. A. Pace, W. R. W a n n e m a c h e r , and H. A. Neufeld, Clin. Chem. 24, 32 (1978). 1~ j. D. M c G a r r y and D. W. Foster, J. Lipid Res. 17, 277 (1976). ~2 R. Parvin and S. V. Pande, Anal. Biochem. 79, 190 (1977).
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Solution B: This contains (in mg/100 ml): choline chloride, 10; biotin, 0.2; calcium pantothenate, 20; thiamine • HCI, 20; pyridoxine • HC1, 20; nicotinic acid, 20; and inositol, 100. It is stored frozen in small aliquots. Carnitine Standard. The standard solution contains per milliliter 0.1 mg of d,/-carnitine, HCI or 50/zg o f / - ( - ) - c a r n i t i n e , HCI in distilled water. The stock solution is diluted 1 : 1000 for a working standard. Preparation of Biological Material for Assay. Extracts can be prepared for assay using methods described by Travassos and Sales ~a or by the methods used to prepare samples for the enzymatic assays described previously in this chapter for total carnitine. The growth responds to /-(-)-carnitine and its derivatives 1s'14 to different extents; thus the carnitine derivatives should be hydrolyzed to free carnitine prior to microbiological assay.
Assay Procedure Double-strength basal medium (2.5 ml) is added to 16 × 125 mm tubes, to which aliquots (0.5, 1.0, 1.5, 2.0, and 2.5 ml) o f unknown samples or standard carnitine (0.001-0.01 tzg/ml) are added; the volume is made to 5.0 ml with distilled water. The tubes are capped and autoclaved. To prepare inocula for assay, the test organism is grown for 24 hr at 37 ° in a screw-capped tube containing single-strength basal medium (5 ml) supplemented with d,/-carnitine (1 mg/liter). The cells are harvested by centrifugation, washed three times by suspension in 5-ml portions of sterile saline 0.9% NaCI, and diluted to an absorbance of 0.14 at 540 nm. The resulting suspension is further diluted 1 : 500 with 0.9% sterile NaC1. Alternatively, the inoculum can be grown on a medium containing (in g/liter): peptone (Difco) 10, glucose, 20, and agar (Difco), 15, at pH 6.5, and then diluted as described previously. One drop of inoculum is added to each tube, which contains 5 ml o f medium plus sample. The tubes are incubated at 37° for 35-48 hr, then shaken on a Vortex mixer; the turbidity is measured at 540 nm. A standard curve of absorbance versus carnitine concentration is used to determine the carnitine content of the unknown. Stock cultures o f the T. bovina mutant are grown at 37 °, for 2 days, on 2% agar slants containing single-strength basal medium supplemented with d,/-carnitine (1 mg/liter); they are transferred at monthly intervals.
Remarks Some biological materials may contain substances that interfere with the assay. This can be determined by measuring the r e c o v e r y of standard i3 L. R. Travassos and C. O. Sales, Anal. Biochem. 58, 485 (1974). ~4 L. M. Lewin and L. L. Bieber, Anal. Biochem. 96, 322 (1979).
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carnitine which has been added to the sample, Travassos and Sales la used ion-exchange treatments to remove interfering substances. Glutamic acid and basic amino acids can promote growth of the mutant, but at much higher concentrations (e.g., 50/xg/ml). Dilution of the material to be assayed may eliminate interference by such compounds.
Isolation, Identification, and Quantitation of Water-Soluble Acylcarnitines Paper Chromatography and Bioautography of l-Carnitine and Its Acyl Esters This sensitive and specific bioautographic method 14is capable of detecting as little as 10 ng of l-carnitine on chromatograms utilizing the carnitine-requiring mutant of Torulopsis bovina.
Reagents The acetyl, propionyl, isobutyryl, and valeryl esters of L-carnitine were prepared by the method of B c h m e r and Bremer. 1~ The basal medium is the same as that described in the section Microbiological Assay for Total l- Carnitine.
Procedure Carnitine and carnitine esters with acyl groups containing 10 carbon atoms or less are dissolved in water. Acylcarnitines with longer chain lengths are dissolved in a mixture of chloroform-methanol, 1 : 1 (v/v), prior to application to Whatman No. 1 filter paper. The papers are developed in the ascending manner using n-propanol-glacial acetic a c i d - H 2 0 , 8 : 1 : 1 (v/v/v), for 16 hr at room temperature or in the descending manner using n-butanol-glacial acetic a c i d - H 2 0 , 8: 1:1 (v/v/v), for 18-24 hr. In order to increase the sensitivity, free carnitine may be liberated from esters in situ by spraying the dried developed chromatograms with concentrated aqueous ammonia, incubating in a humid chamber for 1 hr, and drying prior to bioautographic detection. The test organism is cultured for inoculum as described in the section Microbiological Assay of Total /-Carnitine. The cells are harvested by centrifugation and washed three times with 5 ml of 0.9% NaCI, then are diluted to 100 ml with sterile NaC1. Bioautography agar (150 ml of singlestrength basal medium supplemented with 2% agar) is autoclaved at 121 ° for 15 min, cooled to 45-50 ° in a water bath, inoculated with 0.5 ml of the ~5 T. B c h m e r and J. Bremer, Biochim. Biophys. Acta 152, 559 (1%8).
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inoculum described above, and poured into a sterile rectangular Pyrex baking dish 22 x 34 cm, fitted with an aluminum cover. The bioautograph agar may also be prepared from Yeast Carbon Base (Baltimore Biological Laboratory, Baltimore, Maryland) supplemented with asparagine (1 g/liter) and agar (2%). The chromatograms are placed in contact with the hardened agar surface for 10-15 min. The location of the solvent front and origin is marked on the glass. The sheets are removed and the plates are incubated at 37° for 16-24 hr and then examined, in subdued light, for zones of growth that indicate the regions into which /-carnitine or its derivatives have diffused.
Remarks The Re values of l-carnitine and some of its acylesters in a variety of solvent systems have been reported. TM The two systems recommended here are the most useful for separating acylcarnitines from each other and from carnitine. The descending system completely separates carnitine, acetylcarnitine, and propionylcarnitine. The acyl esters of the volatile acids containing four carbon atoms have greater mobility but are not separated from each other. For example, isobutyryl-, isobutenyl-, and n-butyrylcarnitines are not resolved. The acylcarnitines of 5-carbon acids also move together but are separated from acylcarnitines containing acids of other chain lengths. Solvent systems other than those reported here are needed to separate acylcarnitines of higher molecular weights. The bioautographic method, although useful, is time consuming, and the yeast growth zones are not easy to detect on the agar plates. In addition, some other compounds can give false positive tests. Quantitation of Short-Chain Acylcarnitines by Gas Chromatography
Principle The method depends on separating water-soluble acylcarnitines from other acyl-containing compounds and then saponifying the acylcarnitines and quantitating the individual fatty acids by gas chromatography.
Procedure The method used to separate and quantitate the water-soluble O-acylcarnitines is essentially that described previously TM with major modifications in the steps involving extraction of the volatile fatty acids into ~6 y. R. Choi and L. L. Bieber, Anal. Biochem. 79, 413 (1977).
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diethyl ether prior to gas chromatography. The protocol is outlined here and the modifications will be presented in more detail.
Extraction Procedure 1. Freeze-clamped tissue (both fresh or freeze-clamped tissue are stored at - 72°) is weighed, then minced in 6% HC104 with a ratio of acid to tissue of 5 : 1 (v/w). 2. An internal standard of either of 0.30 mg of crotonylcarnitine or valerylcarnitine is added to the HCIO4 extract. The extract is then thoroughly homogenized with either a Potter-Elvehjem homogenizer or a VirTis homogenizer. 3. The homogenate is centrifuged for 5 min at 12,000g, and the supernatant fluid is decanted and saved. The pellet is washed two times with an amount of 6% HC104 equal to the amount used for the initial homogenization. Each wash is centrifuged for 5 min at 12,000g. The supernatant fluids are combined, and the residue is discarded. 4. The combined extracts are adjusted to pH 6.6 with KOH and allowed to stand on ice for 30 min. After centrifugation at 12,000g for 10 rain, the liquid is decanted and the pellet is washed with 15 ml of absolute ethanol. The sample is centrifuged for 10 rain at 12,000g, and the supernatant fluid is decanted and added to the aqueous supernatant fluid. The combined liquids are evaporated to 5 ml under vacuum using a rotating evaporator. If the tissue samples contain appreciable glycogen, 9 volumes of isopropyl alcohol are added to the supernatant fluid. The samples are allowed to stand on ice for 20 min and then are centrifuged at 12,000g for 5.0 min; the supernatant fluid is saved. This fluid is then evaporated under vacuum to approximately 5 ml to ensure complete removal of isopropyl alcohol and ethanol. The samples can be stored frozen at this point.
Chromatographic Steps Removal of contaminating neutral molecules, cations, and anions involves a series of column chromatographic steps using gel filtration, cationic ion exchange, and anionic ion exchange chromatography. 5. The first column step separates molecules of low molecular weight and of high molecular weight from carnitine and water-soluble acylcarnitines. This is achieved using a BioGel P-2 column with a 2.5 × 45 cm bed volume. The column is poured in a 0.1 mM KH2PO4 buffer and then is calibrated using 10/xl of 0.2 M ADP as the 400 molecular weight marker and radioactive d,/-carnitine for the 200 molecular weight marker. The eluting buffer is 0.1 mM KH2PO4. Elution of ADP is monitored by its ultraviolet absorbance; elution of radioactive carnitine is monitored by
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scintillation counting. When the ADP begins to elute, 70 ml of column effluent are collected, which encompasses the 200-400 molecular weight elution range. After use, the Bio-Gel P-2 column is equilibrated with 0.02% NaN3 to prevent microbial growth. The column is washed thoroughly with 0.1 mM KH2PO4 prior to use. 6. The 70 ml effluent obtained from the Bio-Gel P-2 column (step 5) is then passed through a 2.5 cm x 13 cm Dowex 1 HCOa column ( x 8 , 100200 mesh). The column is eluted with 1.2 bed volumes of distilled water. The effluent is collected in a round-bottom flask and concentrated to 10 ml under vacuum or dried completely by lyophilization and then taken up in 10 ml of water. 7. The pH of the concentrated effluent is adjusted to 2.0 using 1.0 N HCI, and the sample is applied to a Dowex 50 column, 1.0 c m x 12 cm, H + form ( x 8 , 100-200 mesh). The column is thoroughly washed with distilled water, and the water is discarded. The column is then eluted with 0.3 N NH4OH-ethyl alcohol, 8 : 2 (v/v). Pressure of 5 psi can be used to increase the flow rate. As the NH4OH solution passes through the column, a slight color change occurs. When the color change nears the bottom of the column, a 40-ml sample is collected in a graduated cylinder containing 1.0 ml of 1.0 N KOH. The sample is thoroughly mixed and allowed to stand at room temperature for a minimum of 30 rain to saponify the O-acyl derivatives. It is then concentrated to 1 ml with a rotating evaporator under vacuum, and the sample is transferred to a small pearshaped flask (5 or 10 ml). Quantitative transfer is assured by rinsing the round-bottom flask three times with distilled water using 0.5-1.0 ml per rinse. The combined volumes are reduced to 0.5 ml under vacuum. Samples can be stored at this step.
Extraction of Volatile Acids into Ether Step 8 is difficult and is the least reproducible. 8. The sample from step 7 is transferred to a small Teflon screwcapped culture tube, and the flask is rinsed two or three times with 0.5 ml of water. The rinses are added to the culture tube, and a final rinse of 1 ml of diethyl ether is used, which is also added to the culture tube. The samples are placed on ice, and 180/.tl of 6 N HCI are added; the vials are capped tightly and mixed for 1 rain using a Vortex mixer. The samples are then allowed to stand on ice; after the layers have separated, an aliquot of the ether layer is carefully transferred to a 200-/zl Reacti-Vial (Pierce Chemical Company), 20 p.l of 6 N K O H are added, and the sample is vortexed. Most of the ether is then removed from the Reacti-Vial, using a slow stream of nitrogen. After the evaporation, another aliquot of the ether layer from the culture tube is added and the procedure is repeated
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until all the ether has been transferred and evaporated. The ether extraction and evaporation is repeated four times. This rather time-consuming process is necessary in our hands to ensure quantitative transfer of the volatile fatty acids from the acidified saponification mixture to the ether layer. This methodology has allowed us to transfer (recover) more than 90% of the volatile fatty acids from the aqueous phase into the ether phase. The dry samples can be conveniently stored at this stage.
Gas Chromatography 9. The K + salts of the volatile fatty acids must be converted to the free acids and reextracted into ether prior to gas chromatography. This can be accomplished by adding 100 p.l of ether and 25 /xl of 6 N HC1 to the Reacti-Vial. The tightly capped sample is vortexed for 1 min, and the layers are allowed to separate with storage on ice. The ether layer is removed using a Hamilton syringe and transferred to another Reacti-Vial and tightly capped. This ether solution is used for gas chromatography. 16 Good separation of volatile fatty acids has been obtained using 15% SP1220/1% H3PO4 on 100--200 mesh Chromosorb WAW (Supelco) in a 2 mm × 183 cm glass column. Different temperature programs are used depending on the mixture of volatile fatty acids. A temperature increment of 4°/min from 84° to 126° separates most of the mixtures. In lieu of an internal standard, the molar ratios of the various acyl residues can be determined, and these ratios can be used to calculate the amounts of specific acylcarnitines present in the sample if one has determined the total amount of water-soluble acylcarnitines present in tissue as described previously in this chapter.
Remarks The method described above has drawbacks, some of which are listed below. 1. Lack of a suitable internal standard. We have used crotonylcarnitine or n-valerylcarnitine as the internal standard and have obtained excellent results. However, when crotonylcarnitine is used the sample must be processed rapidly, and efforts should be made to minimize oxidation. Unpublished studies have shown that in solution crotonylcarnitine undergoes degradation during standing and processing. Valerylcarnitine has been substituted for crotonylcarnitine, but some tissue samples contain small quantities of valerylcarnitine. This introduces some error in the methodology and also prevents measurement of the amount of valerylcarnitine in the sample. 2. Because of the number of steps and the extraction procedures used it is difficult to process small samples.
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3. The method is adequate only for volatile fatty acids associated with carnitine. If water-soluble acylcarnitines are present that contain nonvolatile carboxylic acids, such as/3-hydroxybutyrate, these would not be detected. 4. It is difficult to establish that the acylcarnitine fraction as isolated is free of other acyl containing compounds; consequently it is always possible that some acyl residues might be derived from compounds other than carnitine. We do not have any evidence that other acyl-containing compounds occur in the final acylcarnitine fraction. The acylcarnitine fraction does not contain compounds such as acylcholines, N-acylamino acids, and neutral molecules. In spite of these limitiations, the methodology can be used for both identification and quantitation of various water-soluble acylcarnitines that may occur in biological tissues. Quantitative Enzymatic Assay of Short-Chain Acylcarnitines after Separation by Paper Chromatography
Principle Esters of carnitine with fatty acids containing 2-5 carbon atoms can be separated by paper chromatography. The location of the acylcarnitine zones can be determined by bioautography of marker strips run alongside the test material. The separated derivatives are then hydrolyzed, and the resulting l-carnitine is eluted and assayed enzymatically as described previously in this chapter. This procedure permits separation and assay of nanomole amounts of acylcarnitines from biological materials.
Reagents Reagents for the enzymatic assay of carnitine are described in the section on assays for carnitine and its derivatives. Acetyl-, propionyl-, butyryl-, and isovalerylcarnitines were synthesized by the method of Bchmer and Bremer. 15
Procedure Test materials (standard acylcarnitines or biological extracts) are applied on a line 8 cm from the edge of a sheet of Whatman No. l paper (16.5 cm x 57 cm) and then are chromatographed, in the descending manner, for 40-48 hr in the solvent system n-butanol-glacial acetic-water, 8 : 1 : 1 (v, v, v). The paper is air-dried; strips containing marker spots are cut from the left and right edges for use in detecting the carnitine-
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containing zones by the bioautographic method, using a carnitinerequiring mutant of the yeast Torulopsis bovina (see section on paper chromatography and bioautography). Using the Re values from the guide strips, the zones containing the separated carnitine derivatives are located and excised. Each zone is moistened with concentrated aqueous ammonia (33%) and incubated at 37° for 1 hr to hydrolyze the acylcarnitines. Carnitine is then eluted from the paper using the method of Esdat and Mirelman. Ir The elution and centrifugation steps are repeated 2 or 3 times; the combined eluates are brought to dryness in an oven at 70° and dissolved in 0.02 M phosphate buffer, pH 7.55, in a volume dependent upon the amount of carnitine present (usually 0.1 ml). The enzymatic method of Cederblad and Lindstedt,s as described above is used to assay the l-carnitine in these fractions. Remarks
Paper chromatography, as described here, separates carnitine and carnitine esters of volatile acids containing 2, 3, 4, and 5 carbons. Recovery of carnitine hydrolyzed from these derivatives is essentially complete (89 to 105%, data unpublished). The method does not distinguish between butyryl-, isobutyryl-, and isobutenylcarnitines, nor between saturated and unsaturated valeryl- and isovalerylcarnitines. Bioautography is used to locate the various carnitine derivatives on guide strips, preparatory to cutting zones for elution and assay, because an alternative method, using iodine staining of the guide strips containing standard compounds (50-100 ng/zone), proved to be unsatisfactory. The mobilities of relatively large amounts of standards were significantly different from the smaller amounts present in the biological samples that were assayed, e.g., 20/xl of human semen. ,r y . E s d a t a n d D. M i r e l m a n , J. Chromatogr. 65, 458 (1972).
[14] D e t e r m i n a t i o n of C h o l i n e , P h o s p h o r y l c h o l i n e , and Betaine By ANTHONY J. BARAK and DEAN J. TUMA
Two methods exist in the literature for the analysis of choline in plasma. The method of Appleton et al. 1 utilizes the formation of choline H. D. A p p l e t o n , B. N. L a D u , Jr., B. B. L e v y , J. M. S t e e l e , a n d B. B. B r o d e , J. Biol.
Chem. 205, 803 (1953).
METHODS IN ENZYMOLOGY, VOL. 72
Copyright © 1981 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181972-8
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Conclusions Many important studies have been conducted with the reductive ozonolysis of fatty acids. Among the different reductive reagents, triphenylphosphine is the most widely used. During reductive ozonolysis, aldehydes, dialdehydes, and aldehyde esters are formed. The aldehyde esters and dialdehydes are less stable than the corresponding acid methyl esters and are usually not commercially available as reference materials. Therefore, these components may need to be synthesized prior to the GLC analyses. The different reductive techniques have undergone a variety of modifications and improvements, but some problems still remain with the GLC analysis of the aldehydic fragments due to chemical residues and also, apparently, problems of overlap between aldehyde and aldehyde esters. Fewer examples of application are as yet available for high-yield oxidative ozonolysis. The method described by the authors is simple and gives excellent yields of the principal acidic products. There is no reason not to apply oxidative ozonolysis in BF3-MeOH to sterols, hydrocarbons, and similar compounds. This method of ozonolysis of methyl esters in BF3-MeOH can be applied directly to monoethylenic and methylene-interrupted di-, tri-, and tetraethylenic fatty acids and apparently also to acetylenic bonds of fatty acids. In the case of non-methyleneinterrupted fatty acids, it became necessary to convert the methyl ester into an alcohol prior to the ozonolysis. When non-methylene-interrupted di-, tri-, and tetraethylenic acids needed to be identified, the authors effected the partial hydrazine reduction of these trienes and tretaenes, isolated the resulting monoethylenic acids, and did the ozonolysis on these simpler fatty acids. It should also be noted that this multiple-operation analysis can give the geometric configuration of particular ethylenic bonds, information not otherwise available for most polyethylenic fatty acids. The combination of BF3-MeOH oxidative ozonolysis and opentubular GLC chromatography on SILAR-5CP, SILAR-7CP, or BDS, which gives an excellent separation of the mono- and diesters, is a powerful tool for structural determination of unsaturated fatty acids.
[13] Measurement of C a r n i t i n e a n d O - A c y l c a r n i t i n e s By L. L. BIEBER and L. M. LEWlN A half century after its isolation from meat, t carnitine was shown to be essential for fl-oxidation of long-chain fatty acids in mammalian sysW. G u l e w i t s c h a n d R. K r i m b e r g ,
METHODS IN ENZYMOLOGY, VOL. 72
Hoppe-Seyler's Z. Physiol. Chem.
45, 326 (1905).
Copyright © 1981 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181972-8
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tems. z,a More recently, the recognition that some human muscle myopathies may be induced by carnitine deficiencies or deficiencies in carnitine acyltransferase activity, and the recognition that carnitine may have more than a single role in intermediary metabolism 4-6 has led to the refinement and development of methods for the measurement of total carnitine and its O-acyl derivatives. This chapter describes some of the newer methodology and some modifications of the older methods but does not repeat the methods described in a previous volume, r
Assay for Free Carnitine, Total Carnitine, and Water-Soluble Acylcarnitines Enzymatic Method Principles
The assays for carnitine are based on the reaction l-Carnitine + acetyl-CoA ~
acetyl-l-carnitine + C o A S H
catalyzed by carnitine acetyltransferase (EC 2.3.1.7). 7 The sensitivity o f the enzymatic method was greatly increased by the introduction of a radioisotopic assay 8 that depends on the incorporation of the acetyl moiety of [1-14C]acetyl-CoA into acetylcarnitine followed by separation and measurement of the amount of [14C]acetylcarnitine formed. The method can be used for free carnitine, total carnitine, and O-acylcarnitines after hydrolysis. The difference between total carnitine and free carnitine yields the amount of O-acylcarnitines. Procedure
Perchloric acid extracts o f weighed tissue are made as described in a previous volume 7 or as described in this chapter in the section on quantitation of acylcarnitines. The extract is divided equally or aliquots are taken; one is neutralized with K O H to precipitate KCIO4, and the others are made 1.0 N with 2 N KOH. The alkaline samples are warmed to 40° for 30 2 I. B. Fritz, Adv. Lipid Res. 1,285 (1963). 3 j. Bremer, J. Biol. Chem. 237, 3628 (1962). 4 M.. E. Mitchell, Am. J. Clin. Nutr. 31, 645 (1978). Y. R. Choi, P. R. H. Clarke, and L. L. Bieber, J. Biol. Chem. 254, 5580 (1979). 6 D. J. Pearson and P. K. Tubbs, Biochem. J. 105, 1953 (1967). r D. J. Pearson, J. F. Chase, and P. K. Tubbs, this series, Vol. 14, p. 612. 8 G. Cederblad and S. Lindstedt, Clin. Chim. Acta 37, 235 (1972).
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min to saponify the short-chain, water-soluble acylcarnitines and then are neutralized with HCIO4. All neutralized samples are chilled for 30 min on ice and then centrifuged to r e m o v e KC104. Aliquots o f the neutralized supernatant fluids are then assayed for carnitine. The protein residue from the perchloric acid extract is suspended in 2 N K O H for 2 hr at 80° and the mixture is then neutralized with HCIO4 and processed as described above. This represents the long-chain acylcarnitine fraction. A modification o f the original C e d e r b l a d - L i n d s t e d t 8 assay is described below. The following stock solutions are made: A: Tris-DTNB containing 0.4 M Tris-HC1, 20 m M D T N B ; p H 7.3 B: Carnitine acetyltransferase; the commercial e n z y m e is diluted 1 : 10 prior to use (0.5 mg/ml) C: [14C]Acetyl-CoA, 3 . 4 5 / z M , 0.2/zCi/ml D: Acetyl-CoA, 0.1 m M Solutions A, C, and D are stored frozen at - 2 0 ° and solution B is made daily from commercial e n z y m e preparations. Prior to assay, two volumes o f solution C are mixed with one volume o f solution A and one volume o f stock solution D to make a total volume sufficient for 100 p.l per assay. After addition o f 100/zl o f assay mixture to 6 x 58 mm glass tubes, then 0, 0.4, 0.8, 1.2, 1.6, and 2.0 nmol of standard l-carnitine (0.10 m M 1-carnitine; 10 /zl/nmol) are added to individual tubes or aliquots o f solutions to be assayed, and distilled water is added to yield a total volume o f 200 p.l. The reaction is started by adding 20 p.l o f e n z y m e solution to make a final volume of 220/.d. Incubations are carried out at 37 ° for 30 min. Next, 200/zl from each tube are pipetted onto a 5 x 35 mm D o w e x 1, C1- column ( 8 x , 200-400 mesh), and the effluent is collected in a scintillation vial. The column is washed twice with 0.5 ml o f distilled water. The entire effluent is counted, and the amount o f carnitine per sample is calculated using the experimentally determined standard curve. Total carnitine = water-soluble (free carnitine) + short-chain (water-soluble acylcarnitine fraction) + long-chain acylcarnitine fraction. Remarks This method provides a measurement o f the total amount of shortchain acylcarnitines, but does not identify the acyl groups. Measurement o f individual acylcarnitines requires separation, identification, and quantitation of the individual components of an acylcarnitine mixture. Methods for both separation and quantitation of the short-chain acylcarnitines are described below. The C e d e r b l a d - L i n d s t e d t assay s has been modified because the presence o f low-molecular-weight short-chain acylcarnitines, such as acetylcarnitine, causes nonlinear standard curves and can cause an overestima-
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tion of the amount of free carnitine in relation to total carnitine by exchange of [14C]acetyl residues from [14C]acetyl-CoA into the acylcarnitine pool. This limitation can be overcome either by increasing the ratio of acetyl-CoA to carnitine 9'~° in order to force the reaction to the right or by adding a reagent to trap free CoA, which pulls the reaction to the right. D T N B 2 sodium tetrathionite H and N-ethylmaleimide ~2 have all been used by different investigators as trapping agents. The method described here uses DTNB, and one of us (L. Lewin) has used the N-ethymalemide modification with good success. Neither of us has used tetrathionate; consequently we do not recommend one method over the other. N-Ethylmaleimide ~2 and tetrathionate H apparently do not inactivate carnitine acetyltransferase as rapidly as DTNB. Microbiological Assay for Total 1-Carnitine
Principle The growth rate of a carnitine-requiring mutant of the yeast Torulopsis bovina can be dependent on the carnitine content of the growth medium when cultures are grown under strictly defined conditions.
Reagents and Organism Assay Organism. A carnitine-requiring strain of the yeast Torupopsis bovina can be obtained from the American Type Culture Collection, Rockville, Maryland (ATTC No. 26014) or from the Centraalbureau voor Schimmelkultures, Delft, Holland (CBS No. 6471). Basal medium. The basal medium is composed of solutions A and B. To prepare double-strength basal medium, solution A (250 ml) and solution B (10 ml) are mixed and brought to a final volume of 500 ml. Solution A: This contains (in g/liter): glucose, 80; L-asparagine - H20, 4.0; KH2PO4, 2.0; MgSO4 7 H20, 2.0; NaC1, 0.4; adenine, 0.04; cytosine, 0.04; oL-tryptophan, 0.20; L-phenylalanine, 0.32; and De-methionine, 0.08. To facilitate solution, the adenine, cytosine, and amino acids are dissolved in a small amount of 1N HCI. Potassium phosphate and magnesium sulfate are dissolved separately and care is taken in mixing the final solution to prevent precipitation of magnesium phosphate. This solution is adjusted to pH 5.0 and stored at 4° under toluene. T. B 0 h m e r , A. Rydning, a n d H. E. Solberg, Clin. Chim. Acta 57, 55 (1974). ~0 j. A. Pace, W. R. W a n n e m a c h e r , and H. A. Neufeld, Clin. Chem. 24, 32 (1978). 1~ j. D. M c G a r r y and D. W. Foster, J. Lipid Res. 17, 277 (1976). ~2 R. Parvin and S. V. Pande, Anal. Biochem. 79, 190 (1977).
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Solution B: This contains (in mg/100 ml): choline chloride, 10; biotin, 0.2; calcium pantothenate, 20; thiamine • HCI, 20; pyridoxine • HC1, 20; nicotinic acid, 20; and inositol, 100. It is stored frozen in small aliquots. Carnitine Standard. The standard solution contains per milliliter 0.1 mg of d,/-carnitine, HCI or 50/zg o f / - ( - ) - c a r n i t i n e , HCI in distilled water. The stock solution is diluted 1 : 1000 for a working standard. Preparation of Biological Material for Assay. Extracts can be prepared for assay using methods described by Travassos and Sales ~a or by the methods used to prepare samples for the enzymatic assays described previously in this chapter for total carnitine. The growth responds to /-(-)-carnitine and its derivatives 1s'14 to different extents; thus the carnitine derivatives should be hydrolyzed to free carnitine prior to microbiological assay.
Assay Procedure Double-strength basal medium (2.5 ml) is added to 16 × 125 mm tubes, to which aliquots (0.5, 1.0, 1.5, 2.0, and 2.5 ml) o f unknown samples or standard carnitine (0.001-0.01 tzg/ml) are added; the volume is made to 5.0 ml with distilled water. The tubes are capped and autoclaved. To prepare inocula for assay, the test organism is grown for 24 hr at 37 ° in a screw-capped tube containing single-strength basal medium (5 ml) supplemented with d,/-carnitine (1 mg/liter). The cells are harvested by centrifugation, washed three times by suspension in 5-ml portions of sterile saline 0.9% NaCI, and diluted to an absorbance of 0.14 at 540 nm. The resulting suspension is further diluted 1 : 500 with 0.9% sterile NaC1. Alternatively, the inoculum can be grown on a medium containing (in g/liter): peptone (Difco) 10, glucose, 20, and agar (Difco), 15, at pH 6.5, and then diluted as described previously. One drop of inoculum is added to each tube, which contains 5 ml o f medium plus sample. The tubes are incubated at 37° for 35-48 hr, then shaken on a Vortex mixer; the turbidity is measured at 540 nm. A standard curve of absorbance versus carnitine concentration is used to determine the carnitine content of the unknown. Stock cultures o f the T. bovina mutant are grown at 37 °, for 2 days, on 2% agar slants containing single-strength basal medium supplemented with d,/-carnitine (1 mg/liter); they are transferred at monthly intervals.
Remarks Some biological materials may contain substances that interfere with the assay. This can be determined by measuring the r e c o v e r y of standard i3 L. R. Travassos and C. O. Sales, Anal. Biochem. 58, 485 (1974). ~4 L. M. Lewin and L. L. Bieber, Anal. Biochem. 96, 322 (1979).
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carnitine which has been added to the sample, Travassos and Sales la used ion-exchange treatments to remove interfering substances. Glutamic acid and basic amino acids can promote growth of the mutant, but at much higher concentrations (e.g., 50/xg/ml). Dilution of the material to be assayed may eliminate interference by such compounds.
Isolation, Identification, and Quantitation of Water-Soluble Acylcarnitines Paper Chromatography and Bioautography of l-Carnitine and Its Acyl Esters This sensitive and specific bioautographic method 14is capable of detecting as little as 10 ng of l-carnitine on chromatograms utilizing the carnitine-requiring mutant of Torulopsis bovina.
Reagents The acetyl, propionyl, isobutyryl, and valeryl esters of L-carnitine were prepared by the method of B c h m e r and Bremer. 1~ The basal medium is the same as that described in the section Microbiological Assay for Total l- Carnitine.
Procedure Carnitine and carnitine esters with acyl groups containing 10 carbon atoms or less are dissolved in water. Acylcarnitines with longer chain lengths are dissolved in a mixture of chloroform-methanol, 1 : 1 (v/v), prior to application to Whatman No. 1 filter paper. The papers are developed in the ascending manner using n-propanol-glacial acetic a c i d - H 2 0 , 8 : 1 : 1 (v/v/v), for 16 hr at room temperature or in the descending manner using n-butanol-glacial acetic a c i d - H 2 0 , 8: 1:1 (v/v/v), for 18-24 hr. In order to increase the sensitivity, free carnitine may be liberated from esters in situ by spraying the dried developed chromatograms with concentrated aqueous ammonia, incubating in a humid chamber for 1 hr, and drying prior to bioautographic detection. The test organism is cultured for inoculum as described in the section Microbiological Assay of Total /-Carnitine. The cells are harvested by centrifugation and washed three times with 5 ml of 0.9% NaCI, then are diluted to 100 ml with sterile NaC1. Bioautography agar (150 ml of singlestrength basal medium supplemented with 2% agar) is autoclaved at 121 ° for 15 min, cooled to 45-50 ° in a water bath, inoculated with 0.5 ml of the ~5 T. B c h m e r and J. Bremer, Biochim. Biophys. Acta 152, 559 (1%8).
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inoculum described above, and poured into a sterile rectangular Pyrex baking dish 22 x 34 cm, fitted with an aluminum cover. The bioautograph agar may also be prepared from Yeast Carbon Base (Baltimore Biological Laboratory, Baltimore, Maryland) supplemented with asparagine (1 g/liter) and agar (2%). The chromatograms are placed in contact with the hardened agar surface for 10-15 min. The location of the solvent front and origin is marked on the glass. The sheets are removed and the plates are incubated at 37° for 16-24 hr and then examined, in subdued light, for zones of growth that indicate the regions into which /-carnitine or its derivatives have diffused.
Remarks The Re values of l-carnitine and some of its acylesters in a variety of solvent systems have been reported. TM The two systems recommended here are the most useful for separating acylcarnitines from each other and from carnitine. The descending system completely separates carnitine, acetylcarnitine, and propionylcarnitine. The acyl esters of the volatile acids containing four carbon atoms have greater mobility but are not separated from each other. For example, isobutyryl-, isobutenyl-, and n-butyrylcarnitines are not resolved. The acylcarnitines of 5-carbon acids also move together but are separated from acylcarnitines containing acids of other chain lengths. Solvent systems other than those reported here are needed to separate acylcarnitines of higher molecular weights. The bioautographic method, although useful, is time consuming, and the yeast growth zones are not easy to detect on the agar plates. In addition, some other compounds can give false positive tests. Quantitation of Short-Chain Acylcarnitines by Gas Chromatography
Principle The method depends on separating water-soluble acylcarnitines from other acyl-containing compounds and then saponifying the acylcarnitines and quantitating the individual fatty acids by gas chromatography.
Procedure The method used to separate and quantitate the water-soluble O-acylcarnitines is essentially that described previously TM with major modifications in the steps involving extraction of the volatile fatty acids into ~6 y. R. Choi and L. L. Bieber, Anal. Biochem. 79, 413 (1977).
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diethyl ether prior to gas chromatography. The protocol is outlined here and the modifications will be presented in more detail.
Extraction Procedure 1. Freeze-clamped tissue (both fresh or freeze-clamped tissue are stored at - 72°) is weighed, then minced in 6% HC104 with a ratio of acid to tissue of 5 : 1 (v/w). 2. An internal standard of either of 0.30 mg of crotonylcarnitine or valerylcarnitine is added to the HCIO4 extract. The extract is then thoroughly homogenized with either a Potter-Elvehjem homogenizer or a VirTis homogenizer. 3. The homogenate is centrifuged for 5 min at 12,000g, and the supernatant fluid is decanted and saved. The pellet is washed two times with an amount of 6% HC104 equal to the amount used for the initial homogenization. Each wash is centrifuged for 5 min at 12,000g. The supernatant fluids are combined, and the residue is discarded. 4. The combined extracts are adjusted to pH 6.6 with KOH and allowed to stand on ice for 30 min. After centrifugation at 12,000g for 10 rain, the liquid is decanted and the pellet is washed with 15 ml of absolute ethanol. The sample is centrifuged for 10 rain at 12,000g, and the supernatant fluid is decanted and added to the aqueous supernatant fluid. The combined liquids are evaporated to 5 ml under vacuum using a rotating evaporator. If the tissue samples contain appreciable glycogen, 9 volumes of isopropyl alcohol are added to the supernatant fluid. The samples are allowed to stand on ice for 20 min and then are centrifuged at 12,000g for 5.0 min; the supernatant fluid is saved. This fluid is then evaporated under vacuum to approximately 5 ml to ensure complete removal of isopropyl alcohol and ethanol. The samples can be stored frozen at this point.
Chromatographic Steps Removal of contaminating neutral molecules, cations, and anions involves a series of column chromatographic steps using gel filtration, cationic ion exchange, and anionic ion exchange chromatography. 5. The first column step separates molecules of low molecular weight and of high molecular weight from carnitine and water-soluble acylcarnitines. This is achieved using a BioGel P-2 column with a 2.5 × 45 cm bed volume. The column is poured in a 0.1 mM KH2PO4 buffer and then is calibrated using 10/xl of 0.2 M ADP as the 400 molecular weight marker and radioactive d,/-carnitine for the 200 molecular weight marker. The eluting buffer is 0.1 mM KH2PO4. Elution of ADP is monitored by its ultraviolet absorbance; elution of radioactive carnitine is monitored by
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scintillation counting. When the ADP begins to elute, 70 ml of column effluent are collected, which encompasses the 200-400 molecular weight elution range. After use, the Bio-Gel P-2 column is equilibrated with 0.02% NaN3 to prevent microbial growth. The column is washed thoroughly with 0.1 mM KH2PO4 prior to use. 6. The 70 ml effluent obtained from the Bio-Gel P-2 column (step 5) is then passed through a 2.5 cm x 13 cm Dowex 1 HCOa column ( x 8 , 100200 mesh). The column is eluted with 1.2 bed volumes of distilled water. The effluent is collected in a round-bottom flask and concentrated to 10 ml under vacuum or dried completely by lyophilization and then taken up in 10 ml of water. 7. The pH of the concentrated effluent is adjusted to 2.0 using 1.0 N HCI, and the sample is applied to a Dowex 50 column, 1.0 c m x 12 cm, H + form ( x 8 , 100-200 mesh). The column is thoroughly washed with distilled water, and the water is discarded. The column is then eluted with 0.3 N NH4OH-ethyl alcohol, 8 : 2 (v/v). Pressure of 5 psi can be used to increase the flow rate. As the NH4OH solution passes through the column, a slight color change occurs. When the color change nears the bottom of the column, a 40-ml sample is collected in a graduated cylinder containing 1.0 ml of 1.0 N KOH. The sample is thoroughly mixed and allowed to stand at room temperature for a minimum of 30 rain to saponify the O-acyl derivatives. It is then concentrated to 1 ml with a rotating evaporator under vacuum, and the sample is transferred to a small pearshaped flask (5 or 10 ml). Quantitative transfer is assured by rinsing the round-bottom flask three times with distilled water using 0.5-1.0 ml per rinse. The combined volumes are reduced to 0.5 ml under vacuum. Samples can be stored at this step.
Extraction of Volatile Acids into Ether Step 8 is difficult and is the least reproducible. 8. The sample from step 7 is transferred to a small Teflon screwcapped culture tube, and the flask is rinsed two or three times with 0.5 ml of water. The rinses are added to the culture tube, and a final rinse of 1 ml of diethyl ether is used, which is also added to the culture tube. The samples are placed on ice, and 180/.tl of 6 N HCI are added; the vials are capped tightly and mixed for 1 rain using a Vortex mixer. The samples are then allowed to stand on ice; after the layers have separated, an aliquot of the ether layer is carefully transferred to a 200-/zl Reacti-Vial (Pierce Chemical Company), 20 p.l of 6 N K O H are added, and the sample is vortexed. Most of the ether is then removed from the Reacti-Vial, using a slow stream of nitrogen. After the evaporation, another aliquot of the ether layer from the culture tube is added and the procedure is repeated
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until all the ether has been transferred and evaporated. The ether extraction and evaporation is repeated four times. This rather time-consuming process is necessary in our hands to ensure quantitative transfer of the volatile fatty acids from the acidified saponification mixture to the ether layer. This methodology has allowed us to transfer (recover) more than 90% of the volatile fatty acids from the aqueous phase into the ether phase. The dry samples can be conveniently stored at this stage.
Gas Chromatography 9. The K + salts of the volatile fatty acids must be converted to the free acids and reextracted into ether prior to gas chromatography. This can be accomplished by adding 100 p.l of ether and 25 /xl of 6 N HC1 to the Reacti-Vial. The tightly capped sample is vortexed for 1 min, and the layers are allowed to separate with storage on ice. The ether layer is removed using a Hamilton syringe and transferred to another Reacti-Vial and tightly capped. This ether solution is used for gas chromatography. 16 Good separation of volatile fatty acids has been obtained using 15% SP1220/1% H3PO4 on 100--200 mesh Chromosorb WAW (Supelco) in a 2 mm × 183 cm glass column. Different temperature programs are used depending on the mixture of volatile fatty acids. A temperature increment of 4°/min from 84° to 126° separates most of the mixtures. In lieu of an internal standard, the molar ratios of the various acyl residues can be determined, and these ratios can be used to calculate the amounts of specific acylcarnitines present in the sample if one has determined the total amount of water-soluble acylcarnitines present in tissue as described previously in this chapter.
Remarks The method described above has drawbacks, some of which are listed below. 1. Lack of a suitable internal standard. We have used crotonylcarnitine or n-valerylcarnitine as the internal standard and have obtained excellent results. However, when crotonylcarnitine is used the sample must be processed rapidly, and efforts should be made to minimize oxidation. Unpublished studies have shown that in solution crotonylcarnitine undergoes degradation during standing and processing. Valerylcarnitine has been substituted for crotonylcarnitine, but some tissue samples contain small quantities of valerylcarnitine. This introduces some error in the methodology and also prevents measurement of the amount of valerylcarnitine in the sample. 2. Because of the number of steps and the extraction procedures used it is difficult to process small samples.
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3. The method is adequate only for volatile fatty acids associated with carnitine. If water-soluble acylcarnitines are present that contain nonvolatile carboxylic acids, such as/3-hydroxybutyrate, these would not be detected. 4. It is difficult to establish that the acylcarnitine fraction as isolated is free of other acyl containing compounds; consequently it is always possible that some acyl residues might be derived from compounds other than carnitine. We do not have any evidence that other acyl-containing compounds occur in the final acylcarnitine fraction. The acylcarnitine fraction does not contain compounds such as acylcholines, N-acylamino acids, and neutral molecules. In spite of these limitiations, the methodology can be used for both identification and quantitation of various water-soluble acylcarnitines that may occur in biological tissues. Quantitative Enzymatic Assay of Short-Chain Acylcarnitines after Separation by Paper Chromatography
Principle Esters of carnitine with fatty acids containing 2-5 carbon atoms can be separated by paper chromatography. The location of the acylcarnitine zones can be determined by bioautography of marker strips run alongside the test material. The separated derivatives are then hydrolyzed, and the resulting l-carnitine is eluted and assayed enzymatically as described previously in this chapter. This procedure permits separation and assay of nanomole amounts of acylcarnitines from biological materials.
Reagents Reagents for the enzymatic assay of carnitine are described in the section on assays for carnitine and its derivatives. Acetyl-, propionyl-, butyryl-, and isovalerylcarnitines were synthesized by the method of Bchmer and Bremer. 15
Procedure Test materials (standard acylcarnitines or biological extracts) are applied on a line 8 cm from the edge of a sheet of Whatman No. l paper (16.5 cm x 57 cm) and then are chromatographed, in the descending manner, for 40-48 hr in the solvent system n-butanol-glacial acetic-water, 8 : 1 : 1 (v, v, v). The paper is air-dried; strips containing marker spots are cut from the left and right edges for use in detecting the carnitine-
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containing zones by the bioautographic method, using a carnitinerequiring mutant of the yeast Torulopsis bovina (see section on paper chromatography and bioautography). Using the Re values from the guide strips, the zones containing the separated carnitine derivatives are located and excised. Each zone is moistened with concentrated aqueous ammonia (33%) and incubated at 37° for 1 hr to hydrolyze the acylcarnitines. Carnitine is then eluted from the paper using the method of Esdat and Mirelman. Ir The elution and centrifugation steps are repeated 2 or 3 times; the combined eluates are brought to dryness in an oven at 70° and dissolved in 0.02 M phosphate buffer, pH 7.55, in a volume dependent upon the amount of carnitine present (usually 0.1 ml). The enzymatic method of Cederblad and Lindstedt,s as described above is used to assay the l-carnitine in these fractions. Remarks
Paper chromatography, as described here, separates carnitine and carnitine esters of volatile acids containing 2, 3, 4, and 5 carbons. Recovery of carnitine hydrolyzed from these derivatives is essentially complete (89 to 105%, data unpublished). The method does not distinguish between butyryl-, isobutyryl-, and isobutenylcarnitines, nor between saturated and unsaturated valeryl- and isovalerylcarnitines. Bioautography is used to locate the various carnitine derivatives on guide strips, preparatory to cutting zones for elution and assay, because an alternative method, using iodine staining of the guide strips containing standard compounds (50-100 ng/zone), proved to be unsatisfactory. The mobilities of relatively large amounts of standards were significantly different from the smaller amounts present in the biological samples that were assayed, e.g., 20/xl of human semen. ,r y . E s d a t a n d D. M i r e l m a n , J. Chromatogr. 65, 458 (1972).
[14] D e t e r m i n a t i o n of C h o l i n e , P h o s p h o r y l c h o l i n e , and Betaine By ANTHONY J. BARAK and DEAN J. TUMA
Two methods exist in the literature for the analysis of choline in plasma. The method of Appleton et al. 1 utilizes the formation of choline H. D. A p p l e t o n , B. N. L a D u , Jr., B. B. L e v y , J. M. S t e e l e , a n d B. B. B r o d e , J. Biol.
Chem. 205, 803 (1953).
METHODS IN ENZYMOLOGY, VOL. 72
Copyright © 1981 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181972-8