ANALYTICAL
BIOCHEMISTRY
A Gas
68, 531-536
(1975)
Chromatographic
L. M.
LEWIN,
A.
Assay PESHIN,
AND
for Carnitine B.
SKLARZ
Department of Chemical Pathology, Sackler Medical School, Tel Aviv University. and Department of Chemistry, Bar I/an University, Ramat Can, Israel
Received February 6, 1975; accepted April 24. 1975 A sensitive gas chromatographic method for the measurement of the biologically significant quaternary ammonium compound, carnitine (P-hydroxy-ytrimethylaminobutyric acid) was developed. based upon decomposition of the compound upon heating at 160°C. in the presence of NaOH and NaBH,. A derivative, present in the chloroform extract of the acidified reaction mixture, was separated on a column of Carbowax 20M (20%) + 3% H,PO, on Chromosorb W, and measured using a He flame ionization detector. The derivative was identified as 4-butyrolactone. based upon its chromatographic retention time and its mass spectrum. Using the reported method, the carnitine concentration of mature rat caput epididymis was 1.9 k 0.5 pmole/g wet wt, while that of cauda epididymis was 16.0 k 1.8 ymolelg wet wt.
L(-)carnitine (P-hydroxy-y-trimethylaminobutyric acid) has long been known as a constituent of animal tissues (l), and plays a role in the transport of fatty acyl groups into mitochondria (2). A possible role for carnitine in sperm maturation is indicated by the recent observations that carnitine is accumulated by bovine sperm during their maturation in the epididymis (3). Marquis and Fritz (4) had previously found that spermatozoa have the highest levels of the enzyme carnitine acetyltransferase, and the epididymis has the highest concentration of carnitine among rat organs. In the past, carnitine has been measured by a number of relatively insensitive biological and chemical assays, which have been reviewed by Fraenkel and Friedman (I), but the currently accepted, sensitive and specific methods are based upon the use of the enzyme carnitine acetyltransferase (5). The gas chromatographic method reported here is an alternative to the enzymatic methods, and is based upon the conversion of carnitine into a volatile product which can be determined by gas chromatography. MATERIALS AND METHODS Materials
D,L-carnitine HCl and acetyl-D,L-carnitine HCI were obtained from Sigma Chemical Co. (St. Louis, MO); palmitoyl-D,L-carnitine HCl from 531 Copyright All rights
0 1975 by Academic Press, Inc. of reproduction in any form reserved.
532
LEWIN,
PESHIN
AND
SKLARZ
Calbiochem (San Diego, CA); and 4-butyrolactone from Merck (Darmstadt, Germany). A sample of palmitoyl-L-carnitine was a generous gift from Professor M. Blecher, Georgetown University School of Medicine, Washington, DC. The Carbowax 20M and Chromosorb W, which were used to prepare the chromatographic columns, were obtained from Supelco, Inc. (Bellefonte, PA), and the columns were prepared by Dr. M. Katz (Packard Instrument Co., Jerusalem, Israel). Methods Gas chromatographic measurement of carnitine. Aqueous samples, or D,L-carnitine HCl standards (IO-50 pg in 0.3 ml) were added to 0.1 ml reagent (2.0 M NaOH containing 40 mg NaBH,/ml) and were heated to dryness at 160” for 30 min. After cooling the sample, 0.5 ml HCl (5.5 M) was added to acidify the mixture and decompose excess NaBH4. The mixture was then extracted with 0.5 ml CHC& and aliquots (5 ~1) of the extract were injected into carrier NZ (20 ml/min) onto a column (6’ X l/s” D) of Carbowax 20M (20%) + 3% H,PO, on Chromosorb W, in a Packard Series 7600, Model 803 Gas Chromatograph (column temperature 115°C inlet and detector temperature 140°C) equipped with a H, flame ionization detector. Concentrations of carnitine in unknown samples were determined by comparing the peak heights with corresponding peaks from the carnitine standard curve. Extraction of carnitine from rat epididymis samples. Mature, male rats of the Charles River strain, obtained from Lowenstein, Yokneam, Israel, were maintained on Purina Lab Chow and water ad lib. After killing the animal by cervical dislocation, the epididymis was excised, cleaned of adhering fat, and dissected into caput, corpus, and caudal portions. The caput and cauda epididimi were weighed, extracted three times by homogenization with 3 ml portions of ethanol (950/o), and centrifuged. Aliquots (1 ml) of the ethanolic solutions were brought to dryness in an oven (50”) prior to addition of reagent for the determination of carnitine by the gas chromatographic method described above. Zdentijication of the carnitine reaction product. An aliquot (25 ~1) of a concentrated chloroform extract containing the derivative produced from carnitine, as described above, was injected into helium carrier gas (20 psi) at 13o”C, onto a column (6 ft X 0.5 cm D), of similar composition to that described above, in a Packard Series 7300, Model 805 gas chromatograph. When the response of the flame ionization detector indicated that the derivative was eluting from the column, a portion of the material was directed into the ionization chamber (temperature 200°C 70 eV), of a Hitachi-Perkin-Elmer Mass Spectrometer (Model RMU 6 E), and the mass spectrum was scanned from 1 to 300 m/e, using an electron multiplier voltage of 1.75 kV, and was corrected for background values.
CHROMATOGRAPHIC
ASSAY
OF
CARNITINE
533
RESULTS
Carnitine, when heated to dryness over an open flame in the presence of NaOH and NaBH,, decomposed, producing a volatile, foul-smelling amine and a residue which could be dissolved in HCl (5.5 M). When aliquots of a chloroform extract of the HCl solution were injected on a gas chromatographic column, as described in Methods, a single peak, with a retention time of 12 min, was well separated from the CHCl, solvent. The product was not found when carnitine was heated with the reagent at 100°C. Suitable conditions for producing the compound reproducibly were investigated, resulting in the conditions described in Methods. Under these conditions the peak heights of this component were found to be proportional to carnitine concentrations, at ieast in the range tested (from 2.5 pg to 1.5 mg). A typical standard curve is shown in Fig. I. Peak heights were found to be proportional to molar concentrations of carnitine, acetylcarnitine, and palmitoylcarnitine, whereas the other biologically significant quaternary ammonium compounds tested (choline and betaine), did not produce this derivative. The nature of the volatile product was determined by mass spectrometry of the material in the gas chromatographic peak. Thermal decomposition of carnitine, under the basic conditions of our experiment might be expected to yield a compound related to a four carbon carboxylic acid (1). The relative abundances of the components, obtained 70-
60-
0
25
50 Carnitine.HCI (WI
75
400
FIG. 1. Standard curve for gas chromatographic analysis of carnitine. D,L-carnitine HCl standards containing 25, SO, 75, and 100 pg of carnitine HCl were reacted, extracted, and aliquots (5 ~1) of the CHCIR phase were injected onto a gas chromatographic column, as described in Methods. The recorded peak heights were obtained using the HP flame ionization detector described, with electrometer sensitivity of lo-” amp.
534
LEWIN,
PESHIN
AND
TABLE MASS
SPECTRA
SKLARZ
1
OF SOME BUTYRIC ACID DERIVATIVES CARNITINE REACTION PRODLJC~
AND
OF THE
Relative abundance
m/e 37 38 39 41 42 45 56 58 68 69 85 86
Carnitine derivativea
4-Butyrolactonea
4-ButyrolactoneC
36 100 200
44 100 176
34 100 218
58
62
68
Vinylacetic acidc 153 24 107 100 51 32
CrotonicacidC
Isocrotonicacid”
Methylacrylic acidC
21 94 100 17 26
24 35 144 100 33 34
29 36
61 34
14
101
76
71
14 65 100 84 14
27 20 68
19 79
52
26
D Mass spectra of 4-butyrolactone and of the carnitine reaction product were by combined gas chromatography-mass spectrometry, as described in Methods. maining data was obtained from Cornu and Massot (6). AU data is reported in relative peak heights, with the value of peak height at m/e = 41 being set at the peak heights at other mass values being related thereto. a Experimental data, obtained as described in Methods. c Literature values (6).
obtained The reterms of 100, and
by mass spectrometry, are shown in Table 1. Comparison of the mass of the heaviest component in the mass spectrum (Mass = 86), with that of butyric acid (Mass = 88), indicated the possibility that the product might be an unsaturated or cyclized derivative of butyric acid. Comparison of the spectrum which we obtained (Table 1) with those reported (6) for unsaturated, four carbon acids and for 4-butyrolactone indicated that the lactone was the likely structure. This was confirmed when standard 4butyrolactone, injected on the chromatographic columns under similar conditions, produced an essentially identical mass spectrum, and eluted at an identical retention time as did the unknown derivative. The metastable ion, which would be predicted to appear upon the loss of CO, from the butyrolactone, was found (Mass = 20.5) in the spectrum of the authentic lactone and of the carnitine derivative. DISCUSSION
Carnitine, like other quaternary ammonium compounds, is highly polar and nonvolatile. In order to prepare a derivative suitable for gas chromatography, carnitine was reacted with NaOH at high temperature,
CHROMATOGRAPHIC
ASSAY
OF
535
CARNITINE
under conditions similar to those of the Hofmann degradation, which is known to decompose certain quaternary ammonium compounds, with release of trimethylamine and an additional fragment characteristic of the amine in question (8). Under the conditions used, carnitine produced a derivative which was shown to have a retention time and mass spectrum corresponding to those of 4-butyrolactone. The amount of this material which was produced was proportional to the amount of carnitine present. Acetylcarnitine and palmitoylcarnitine, which would be expected to be hydrolyzed to carnitine under the conditions used, produced peaks, as predicted from their carnitine content. Betaine and choline did not produce peaks on the chromatogram. The formation of 4-butyrolactone presumably involves CYp-elimination, lactonisation in the allylic ammonium compound formed, and borohydride reduction of the a&double bond: Me,--l;-CH,-CH(OH)--CH,CO, -
OH-
Me,--I;-CH,-
CH=CHCO;
1 OH-
0
0
The gas chromatographic method reported here requires inexpensive reagents, is sensitive and rapid, and presents a practical and useful alternative to enzymatic methods currently in use. Since the gas chromatographic method measures total carnitine, while the enzymatic method, using carnitine acetyl transferase, is specific for free carnitine, the methods are complementary. A possible source of error in the determination of carnitine, in natural materials, by the gas chromatographic method, would be the presence of butyrolactone, or compounds such as crotonic acid, which might produce it. In order to establish the utility of the method for measurement of carnitine in reproductive tissue, the carnitine concentration of the cauda epididymis (16.0 * I .8 pmole/g wet wt) and the caput epididymis (1.9 -+ 0.5 pmole/g wet wt) of the mature, male rat, were measured, as described in Methods. The close agreement between these results and these of Brooks et al. (7), for mature, male rats of the same strain (caput epididymis 2.30 & 0.26; cauda epididymis plus ductus deferens 15.3 2 1.5), who used an enzymatic carnitine determination, indicated that the gas chromatographic method is suitable for assaying carnitine in this material. The gas chromatographic method was routinely used to measure 25-100 pg of carnitine, and increased sensitivity (to 2.5 kg) was achieved by using smaller volumes of reagent and chloroform. This is comparable to the sensitivities reported (5) for the enzymatic
536
LEWIN,
PESHIN AND SKLARZ
method (approximately 10 nmole, equivalent to 2 pg). If the gas chromatographic procedure is to be used to measure total carnitine in samples which contain long-chain acylcarnitines as well as short-chain acylcarnitines and free carnitine, a method suitable for quantitative extraction of all of these compounds should be chosen (9, 10). ACKNOWLEDGMENTS The authors gratefully acknowledge stimulating discussions with Dr. Michael Katz, and the skilled technical assistance of Mr. Amos Gabso. This work was supported, in part, by a grant to Professor L. M. Lewin, from the Ministry of Health, Government of Israel, and by grant no. 6700470, from the Ford Foundation to Professor B. Lunenfeld, of the Institute of Endocrinology, Chaim Sheba Medical Center, Tel Hashomer, Israel.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
Fraenkel, B., and Friedman, S. (1957) Vitam. Horn 15, 73-118. Fritz, I. B. (1963) Adv. Lipid Res. 1, 285-334. Casillas, E. (1973) J. Biol. Chem. 248, 8227-8232. Marquis, N. R., and Fritz, 1. B. (1965) J. Biol. Chem. 240, 2 197-2200. Pearson, D. J., Chase, J. F. A., and Tubbs, P. K. (1969) in Methods in Enzymology (Lowenstein, J. M., ed.) Vol. 14, pp. 612-622, Academic Press, New York. Cornu. A., and Massot, R. (1966) Compilation of Mass Spectral Data, p. 14C, Heyden and Sons, London. Brooks, D. E., Hamilton, D. W., and Mallek, A. H. ( 1974) J. Reprod. Ferf. (1974) 36, 141-160. Spialter, L., and Pappalardo, J. A. (1968) The Cyclic Aliphatic Tertiary Amines, p. 74, MacMillan Co., New York. Pearson, D. J.. and Tubbs, P. K. (1964) Biochim. Biophys. Acta 84, 772-773. Mehlman, M. A., and Therriault, D. G. (1965) in Recent Research on Carnitine: Its Relationship to Lipid Metabolism. (Wolf, G. ed.) pp. 35-43, M.I.T. Press, Cambridge, MA.
BIOCHEMISTRY
A Gas
68, 531-536
(1975)
Chromatographic
L. M.
LEWIN,
A.
Assay PESHIN,
AND
for Carnitine B.
SKLARZ
Department of Chemical Pathology, Sackler Medical School, Tel Aviv University. and Department of Chemistry, Bar I/an University, Ramat Can, Israel
Received February 6, 1975; accepted April 24. 1975 A sensitive gas chromatographic method for the measurement of the biologically significant quaternary ammonium compound, carnitine (P-hydroxy-ytrimethylaminobutyric acid) was developed. based upon decomposition of the compound upon heating at 160°C. in the presence of NaOH and NaBH,. A derivative, present in the chloroform extract of the acidified reaction mixture, was separated on a column of Carbowax 20M (20%) + 3% H,PO, on Chromosorb W, and measured using a He flame ionization detector. The derivative was identified as 4-butyrolactone. based upon its chromatographic retention time and its mass spectrum. Using the reported method, the carnitine concentration of mature rat caput epididymis was 1.9 k 0.5 pmole/g wet wt, while that of cauda epididymis was 16.0 k 1.8 ymolelg wet wt.
L(-)carnitine (P-hydroxy-y-trimethylaminobutyric acid) has long been known as a constituent of animal tissues (l), and plays a role in the transport of fatty acyl groups into mitochondria (2). A possible role for carnitine in sperm maturation is indicated by the recent observations that carnitine is accumulated by bovine sperm during their maturation in the epididymis (3). Marquis and Fritz (4) had previously found that spermatozoa have the highest levels of the enzyme carnitine acetyltransferase, and the epididymis has the highest concentration of carnitine among rat organs. In the past, carnitine has been measured by a number of relatively insensitive biological and chemical assays, which have been reviewed by Fraenkel and Friedman (I), but the currently accepted, sensitive and specific methods are based upon the use of the enzyme carnitine acetyltransferase (5). The gas chromatographic method reported here is an alternative to the enzymatic methods, and is based upon the conversion of carnitine into a volatile product which can be determined by gas chromatography. MATERIALS AND METHODS Materials
D,L-carnitine HCl and acetyl-D,L-carnitine HCI were obtained from Sigma Chemical Co. (St. Louis, MO); palmitoyl-D,L-carnitine HCl from 531 Copyright All rights
0 1975 by Academic Press, Inc. of reproduction in any form reserved.
532
LEWIN,
PESHIN
AND
SKLARZ
Calbiochem (San Diego, CA); and 4-butyrolactone from Merck (Darmstadt, Germany). A sample of palmitoyl-L-carnitine was a generous gift from Professor M. Blecher, Georgetown University School of Medicine, Washington, DC. The Carbowax 20M and Chromosorb W, which were used to prepare the chromatographic columns, were obtained from Supelco, Inc. (Bellefonte, PA), and the columns were prepared by Dr. M. Katz (Packard Instrument Co., Jerusalem, Israel). Methods Gas chromatographic measurement of carnitine. Aqueous samples, or D,L-carnitine HCl standards (IO-50 pg in 0.3 ml) were added to 0.1 ml reagent (2.0 M NaOH containing 40 mg NaBH,/ml) and were heated to dryness at 160” for 30 min. After cooling the sample, 0.5 ml HCl (5.5 M) was added to acidify the mixture and decompose excess NaBH4. The mixture was then extracted with 0.5 ml CHC& and aliquots (5 ~1) of the extract were injected into carrier NZ (20 ml/min) onto a column (6’ X l/s” D) of Carbowax 20M (20%) + 3% H,PO, on Chromosorb W, in a Packard Series 7600, Model 803 Gas Chromatograph (column temperature 115°C inlet and detector temperature 140°C) equipped with a H, flame ionization detector. Concentrations of carnitine in unknown samples were determined by comparing the peak heights with corresponding peaks from the carnitine standard curve. Extraction of carnitine from rat epididymis samples. Mature, male rats of the Charles River strain, obtained from Lowenstein, Yokneam, Israel, were maintained on Purina Lab Chow and water ad lib. After killing the animal by cervical dislocation, the epididymis was excised, cleaned of adhering fat, and dissected into caput, corpus, and caudal portions. The caput and cauda epididimi were weighed, extracted three times by homogenization with 3 ml portions of ethanol (950/o), and centrifuged. Aliquots (1 ml) of the ethanolic solutions were brought to dryness in an oven (50”) prior to addition of reagent for the determination of carnitine by the gas chromatographic method described above. Zdentijication of the carnitine reaction product. An aliquot (25 ~1) of a concentrated chloroform extract containing the derivative produced from carnitine, as described above, was injected into helium carrier gas (20 psi) at 13o”C, onto a column (6 ft X 0.5 cm D), of similar composition to that described above, in a Packard Series 7300, Model 805 gas chromatograph. When the response of the flame ionization detector indicated that the derivative was eluting from the column, a portion of the material was directed into the ionization chamber (temperature 200°C 70 eV), of a Hitachi-Perkin-Elmer Mass Spectrometer (Model RMU 6 E), and the mass spectrum was scanned from 1 to 300 m/e, using an electron multiplier voltage of 1.75 kV, and was corrected for background values.
CHROMATOGRAPHIC
ASSAY
OF
CARNITINE
533
RESULTS
Carnitine, when heated to dryness over an open flame in the presence of NaOH and NaBH,, decomposed, producing a volatile, foul-smelling amine and a residue which could be dissolved in HCl (5.5 M). When aliquots of a chloroform extract of the HCl solution were injected on a gas chromatographic column, as described in Methods, a single peak, with a retention time of 12 min, was well separated from the CHCl, solvent. The product was not found when carnitine was heated with the reagent at 100°C. Suitable conditions for producing the compound reproducibly were investigated, resulting in the conditions described in Methods. Under these conditions the peak heights of this component were found to be proportional to carnitine concentrations, at ieast in the range tested (from 2.5 pg to 1.5 mg). A typical standard curve is shown in Fig. I. Peak heights were found to be proportional to molar concentrations of carnitine, acetylcarnitine, and palmitoylcarnitine, whereas the other biologically significant quaternary ammonium compounds tested (choline and betaine), did not produce this derivative. The nature of the volatile product was determined by mass spectrometry of the material in the gas chromatographic peak. Thermal decomposition of carnitine, under the basic conditions of our experiment might be expected to yield a compound related to a four carbon carboxylic acid (1). The relative abundances of the components, obtained 70-
60-
0
25
50 Carnitine.HCI (WI
75
400
FIG. 1. Standard curve for gas chromatographic analysis of carnitine. D,L-carnitine HCl standards containing 25, SO, 75, and 100 pg of carnitine HCl were reacted, extracted, and aliquots (5 ~1) of the CHCIR phase were injected onto a gas chromatographic column, as described in Methods. The recorded peak heights were obtained using the HP flame ionization detector described, with electrometer sensitivity of lo-” amp.
534
LEWIN,
PESHIN
AND
TABLE MASS
SPECTRA
SKLARZ
1
OF SOME BUTYRIC ACID DERIVATIVES CARNITINE REACTION PRODLJC~
AND
OF THE
Relative abundance
m/e 37 38 39 41 42 45 56 58 68 69 85 86
Carnitine derivativea
4-Butyrolactonea
4-ButyrolactoneC
36 100 200
44 100 176
34 100 218
58
62
68
Vinylacetic acidc 153 24 107 100 51 32
CrotonicacidC
Isocrotonicacid”
Methylacrylic acidC
21 94 100 17 26
24 35 144 100 33 34
29 36
61 34
14
101
76
71
14 65 100 84 14
27 20 68
19 79
52
26
D Mass spectra of 4-butyrolactone and of the carnitine reaction product were by combined gas chromatography-mass spectrometry, as described in Methods. maining data was obtained from Cornu and Massot (6). AU data is reported in relative peak heights, with the value of peak height at m/e = 41 being set at the peak heights at other mass values being related thereto. a Experimental data, obtained as described in Methods. c Literature values (6).
obtained The reterms of 100, and
by mass spectrometry, are shown in Table 1. Comparison of the mass of the heaviest component in the mass spectrum (Mass = 86), with that of butyric acid (Mass = 88), indicated the possibility that the product might be an unsaturated or cyclized derivative of butyric acid. Comparison of the spectrum which we obtained (Table 1) with those reported (6) for unsaturated, four carbon acids and for 4-butyrolactone indicated that the lactone was the likely structure. This was confirmed when standard 4butyrolactone, injected on the chromatographic columns under similar conditions, produced an essentially identical mass spectrum, and eluted at an identical retention time as did the unknown derivative. The metastable ion, which would be predicted to appear upon the loss of CO, from the butyrolactone, was found (Mass = 20.5) in the spectrum of the authentic lactone and of the carnitine derivative. DISCUSSION
Carnitine, like other quaternary ammonium compounds, is highly polar and nonvolatile. In order to prepare a derivative suitable for gas chromatography, carnitine was reacted with NaOH at high temperature,
CHROMATOGRAPHIC
ASSAY
OF
535
CARNITINE
under conditions similar to those of the Hofmann degradation, which is known to decompose certain quaternary ammonium compounds, with release of trimethylamine and an additional fragment characteristic of the amine in question (8). Under the conditions used, carnitine produced a derivative which was shown to have a retention time and mass spectrum corresponding to those of 4-butyrolactone. The amount of this material which was produced was proportional to the amount of carnitine present. Acetylcarnitine and palmitoylcarnitine, which would be expected to be hydrolyzed to carnitine under the conditions used, produced peaks, as predicted from their carnitine content. Betaine and choline did not produce peaks on the chromatogram. The formation of 4-butyrolactone presumably involves CYp-elimination, lactonisation in the allylic ammonium compound formed, and borohydride reduction of the a&double bond: Me,--l;-CH,-CH(OH)--CH,CO, -
OH-
Me,--I;-CH,-
CH=CHCO;
1 OH-
0
0
The gas chromatographic method reported here requires inexpensive reagents, is sensitive and rapid, and presents a practical and useful alternative to enzymatic methods currently in use. Since the gas chromatographic method measures total carnitine, while the enzymatic method, using carnitine acetyl transferase, is specific for free carnitine, the methods are complementary. A possible source of error in the determination of carnitine, in natural materials, by the gas chromatographic method, would be the presence of butyrolactone, or compounds such as crotonic acid, which might produce it. In order to establish the utility of the method for measurement of carnitine in reproductive tissue, the carnitine concentration of the cauda epididymis (16.0 * I .8 pmole/g wet wt) and the caput epididymis (1.9 -+ 0.5 pmole/g wet wt) of the mature, male rat, were measured, as described in Methods. The close agreement between these results and these of Brooks et al. (7), for mature, male rats of the same strain (caput epididymis 2.30 & 0.26; cauda epididymis plus ductus deferens 15.3 2 1.5), who used an enzymatic carnitine determination, indicated that the gas chromatographic method is suitable for assaying carnitine in this material. The gas chromatographic method was routinely used to measure 25-100 pg of carnitine, and increased sensitivity (to 2.5 kg) was achieved by using smaller volumes of reagent and chloroform. This is comparable to the sensitivities reported (5) for the enzymatic
536
LEWIN,
PESHIN AND SKLARZ
method (approximately 10 nmole, equivalent to 2 pg). If the gas chromatographic procedure is to be used to measure total carnitine in samples which contain long-chain acylcarnitines as well as short-chain acylcarnitines and free carnitine, a method suitable for quantitative extraction of all of these compounds should be chosen (9, 10). ACKNOWLEDGMENTS The authors gratefully acknowledge stimulating discussions with Dr. Michael Katz, and the skilled technical assistance of Mr. Amos Gabso. This work was supported, in part, by a grant to Professor L. M. Lewin, from the Ministry of Health, Government of Israel, and by grant no. 6700470, from the Ford Foundation to Professor B. Lunenfeld, of the Institute of Endocrinology, Chaim Sheba Medical Center, Tel Hashomer, Israel.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
Fraenkel, B., and Friedman, S. (1957) Vitam. Horn 15, 73-118. Fritz, I. B. (1963) Adv. Lipid Res. 1, 285-334. Casillas, E. (1973) J. Biol. Chem. 248, 8227-8232. Marquis, N. R., and Fritz, 1. B. (1965) J. Biol. Chem. 240, 2 197-2200. Pearson, D. J., Chase, J. F. A., and Tubbs, P. K. (1969) in Methods in Enzymology (Lowenstein, J. M., ed.) Vol. 14, pp. 612-622, Academic Press, New York. Cornu. A., and Massot, R. (1966) Compilation of Mass Spectral Data, p. 14C, Heyden and Sons, London. Brooks, D. E., Hamilton, D. W., and Mallek, A. H. ( 1974) J. Reprod. Ferf. (1974) 36, 141-160. Spialter, L., and Pappalardo, J. A. (1968) The Cyclic Aliphatic Tertiary Amines, p. 74, MacMillan Co., New York. Pearson, D. J.. and Tubbs, P. K. (1964) Biochim. Biophys. Acta 84, 772-773. Mehlman, M. A., and Therriault, D. G. (1965) in Recent Research on Carnitine: Its Relationship to Lipid Metabolism. (Wolf, G. ed.) pp. 35-43, M.I.T. Press, Cambridge, MA.
