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Analysis of isethionic acid in mammalian tissues
Life Sciences Vol . 20, pp . 2029-2036, 1977 Printed In The U.S .A .
Pergamon Press
ANALYSIS OF ISETHIONIC ACID IN MAMMALIAN TISSUES Mohamed A . Remtulla, Derek A . Applegarth, Donald G . Clark and Ian H . Williams Departments of Paediatrics, Pathology and Chemistry, The University of British Columbia, Biochemical Diseases Laboratory, Children's Hospital and Government of Canada Agriculture Research Station, Vancouver, British Columbia (Received in final form May 17, 1977) SUMMARY
A gas-liquid chromatographic assay has been developed to measure isethionic acid after methylation with diazomethane . The identity of the products o£ methylation has been confirmed by mass-spectrometry and nuclear magnetic resonance spectroscopy . The method was used to measure isethionic acid in rat heart, dog heart and rat brain . The assay was validated by measuring isethionic acid on squid axoplasm . We have been able to detect only trace amounts of isethionic acid in rat brain (0 .2 mg/1008) and rat heart (0 .1 mg/ 1008) . None was found in dog heart . Isethionic acid, 2-hydroxy-ethane sulfonic acid, is the deaminated analogue of Its presence in biological material was first reported by taurine (1) . Roechlin (2) who found that it was the major anion of the axoplasm from the squid giant axon . He suggested that ISA might indirectly be responsible for the production of electrical phenomena in the nerve . Welty et al . (3) proposed that taurine was the precursor of ISA. These workers apparently isolated substantial quantities of ISA from dog and rat heart tissues, by a gravimetric method involving the crystallization of ISA as its sodium salt from a hot aqueous extract of dog heart or rat heart . They later demonstrated the conversion of 35 S-taurine to 35 S-ISA by dog heart slices (4) . Later Peck and Awapara (5) reported that very small amounts of isethionic acid could be formed from isotopically labelled taurine in rat heart and brain tissues . Other workers (6,7) have suggested that the conversion of taurine to ISA in myocardial cells facilitates the retention of intracellular calcium or potassium ions . The major difficulty of studying the function of ISA has been the lack of a good analytical procedure for its detection and quantitation . Methods used in the past to detect ISA do not offer much accuracy or sensitivity and some apparently promising methods have never been published in full (8,9) . We therefore report here an analytical method to measure ISA. MATERIALS AND METHODS For analysis of rat tissues, we used Wistar rats weighing approximately 200g . Animals were sacrificed by a sharp blow to the head . Hearts and brains were promptly excised and the tissues rinsed in normal saline, blotted on Wha,man Correspondence to : Derek A. Applegarth, Biochemical Diseases Laboratory, Children's Hospital, 250 West 59th Avenue, Vancouver, B.C . Canada V5X 1X2 . 2029
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#1 filter paper, and immediately frozen in small plastic vials in liquid nitrogen . This process required less than ten minutes per rat . For analysis of dog heart, animals were obtained from the Department of Physiology at The University of British Columbia, after having been used for open heart surgery . The dogs were sacrificed with 15% potassium chloride (10 ml) and the heart stored at -20 degrees before use . Samples of the giant axon of squid (Loligo pealii) were obtained from Dr . F .C .G . Hoskin of the Department of Biology, Illinois Institute of Technology, Chicago, Illinois . Isolation of ISA from Heart and Brain Tissues : 5g samples of pooled brain or heart tissues were used for experimentation . The heart tissue was minced before being used and then divided into two equal portions of 2 .5g each . To one portion was added 1 .0 ymole of sodium isethionate (Sigma Chemical Company, St . Louis, Missouri) . The other portion was used without any addition . Both portions were homogenized in 50% methanol/water (v/v), (10 ml), in a Sorvall omnimixer (Ivan Sorvall Inc ., Norwalk, Connecticut), using a teflon chamber at 3/4 of the full speed for five minutes, followed by one minute of full speed. The homogenate was transferred to a centrifuge tube and centrifuged at 3,000g for five minutes . The homogenizing chamber was rinsed three times with 5 ml, 50% methanol/water (v/v), and the rinse washings added to the centrifuge pellet which was suspended in the rinsing solution using a Vortex mixer . The resulting suspension was then centrifuged again for five minutes and the supernatant fluid removed . To the combined volume of supernatants and rinsings, an equal volume of Folch solvent (chloroform:methanol, 2:1, v/v) was added . The solutions were mixed thoroughly and centrifuged to separate the layers . The upper aqueous layer was removed and evaporated to dryness in a rotary evaporator under reduced pressure . After evaporation of the aqueous layer to dryness, 2 ml of a purified cation exchange resin (AG-50W-X8, 50-100 mesh, H+ form Bio Rad Laboratories, Richmond, California), prewashed in methanol and suspended in an equal volume of methanol, was added to the flask. After trituration, the mixture was transferred to a 5 ml glass-stoppered conical centrifuge tube . After centrifugation, the methanol layer was dried in a vacuum desiccator over sulphuric acid . Isolation of ISA from Axoplasm of the Squid Giant Axon : Axoplasm from the squid giant axon was obtained in freeze-dried form . The sample, 83mg fresh weight axoplasm, was dispersed in deionized water using a glass homogenizer . The turbid solution so formed was made up to a volume of 10 ml with deionized water . An aliquot of the solution was mixed with an equal volume of absolute methanol and this mixture treated with an equal volume of Folch solvent . The rest of the procedure was the same as that described above, for isolation of ISA from heart and brain tissues . Preparation of Standards : A suspension of ion exchange resin AG-50 (H+ form in methanol, 1 ml of resin + 1 ml of methanol) was pipetted into each of six, 5 ml glass stoppered, calibrated, conical centrifuge tubes . After allowing the resin to settle, the methanol layer was aspirated and discarded . To each tube was added 0, 0.1, 0 .2, 0.3, 0 .4, 0.5 and 0 .6 ml aliquots of 5 mM sodium isethionate solution in water. Volume was made up to 4 ml in each tube with methanol, and the resin suspended using a Vortex mixer. After centrifugation, the methanol layer from each tube was removed into reaction vials and evaporated to dryness in a vacuum desiccator over sulphuric acid ._ Abbreviations : Throughout the text, isethionic acid is abbreviated as ISA. Nuclear magnetic resonance spectroscopy is abbreviated as NMR .
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Methylation of the Samples and Preparation of a Standard Curve : ISA standards, or samples obtained from a tissue extract, were dissolved in 0 .1 ml of a 1 MM solution of salicylic acid (Sigma Chemical Company, St . Louis, Missouri) in methanol . Salicylic acid was used as an internal standard . The vials were stoppered using teflon laminated discs and placed in an ice-bath for five minutes . Ethereal diazomethane solution prepared from diazald (Aldrich Chemical Company, Milwaukee, Wisconsin) was introduced into the vials slowly, with mixing until the yellow color persisted . An additional three drops of diazomethane solution was added and the mixture was allowed to stand for about thirty minutes The total volume in the vial was 0.2 ml or less . Where the total in ice . volume in the vial was more than 0.2 ml, the excess solvent was carefully evaporated under a stream of dry nitrogen and the sample remethylated for a further thirty minute period . Methylation reactions were all stopped by adding one drop 50% acetic acid in water (v/v) . This remethylation procedure was found not to affect the standard calibration curve for ISA . The gas-liquid chromatoGas-Liquid Chromatography : Flame Ionization Detection : graph used was a Bendix,Model 2500, equipped with a flame ionization detector . Columns were 6 ft by 4 mm (i .d .) glass U-tubes . The stationary phases used were 5% OV-1 and 5% OV-17 (Gas Chromatographic Specialities Ltd . ; Brockville, Ontario) on 80-100 mesh H.P . Chromosorb W. Analyses were performed isothermally at temperatures ranging from 100 degrees C to 150 degrees C . The optimal temperature for OV-1 was 115 degrees C and for OV-17 was 135 degrees C . Nitrogen was used as a carrier gas at a flow rate of 40 ml/min . Gas-Liquid Chromatography : Sulphur Detection : The gas-liquid chromatograph used for such experiments was a Micro Tek 220 equipped with a flame photometric detector, Model FPD 100 (Melpar Inc ., Falls Church, Virginia) . The column used was a~6 ft by 2 mm (i .d .) glass U-tube with 5% OV-17 on 80-100 mesh H.P . Chromosorb W . Nitrogen was used as the carrier gas with an inlet flow of 30 ml/min . Column temperature was 100 degrees C . Oxygen flow to the detector was 10 ml/min hydrogen flow was 70 ml/min and air flow was 30 ml/min . Gas-Liquid Chromatography : Mass Spectrometry : The mass-spectrometer used was a Hitachi Perkin-Elmer, operating at an ionization energy of 70 ev . interfaced with a Varian, Model 1400 gas chromatograph . Authentic ISA and butane sulfonic acid samples were analyzed on OV-17 column after methylation with diazomethane . Chromatographic details were as outlined above for flame ionization detection . Nuclear Magnetic Resonance Spectroscopy : For proton NMR spectroscopy, the methyl esters of ISA and butane sulfonic acid were prepared in the following manner . Five times recrystallized sodium isethionate (100 mg) in boiling absolute ethanol and 1-butanesulfonic acid sodium salt (100 mg) (Eastman Kodak Company, Rochester, N.Y .) were each dissolved in methanol (1 .0 ml) and the samples treated with resin, and methylated as described above . After thirty minutes, excess diazomethane was carefully blown off with a stream of dry nitrogen and the sample completely dried down under reduced pressure over sulphuric acid . The NMR spectra of the reaction products, without further purification, were taken at 100 mHz in a Varian HA-100 spectrometer . Samples were dissolved in deuterated dimethylsulfoxide (Merck, Sharpe and Dohme Ltd., Canada) with tetramethyl silane being used as an internal standard . For the purposes of comparison, the NMR spectrum of crystallized, non-methylated ISA, also dissolved in dimethylsulfoxide was obtained . RESULTS Chromatography of the methylated ISA on OV-1 and on an OV-17 column is shown in Figure 1. Using a column of 5% OV-1, a single peak with a retention time of 1 .6 minutes was obtained for methylated ISA . Using a column of 5% OV-17, two
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peaks at retention times of 3 .5 and 4 .0 minutes were obtained . The identity of these two peaks was established by the use of gas chromatography mass-spectrometry (Figure 2) and nuclear magnetic resonance spectroscopy . On OV-17 the peak area of the large peak was approximately 20 times that of the smaller peak . Interpretation of the mass-spectrometry fragmentation pattern of the two peaks obtained after OV-17 gas chromatography is shown in Figure 2 . The large peak (Peak II) was the methylester of the ISA, while the small peak (Peak I) was the methylether, methylester of ISA.
B
A
~Solvent Peak
C
f- Solvent Peak
"--- Peak
Peak II 11
Isethionic Acid
Salicylic Acid Peak
1
Salicylic Acid
FIG . 1 Chromatographic separation of the products of methylation of isethionic and salicylic acid using flame ionization detection . A. The column used was a 5% OV-1 column ; oven temperature 115 degrees B. The column used was a 5%OV-17 column ; oven temperature 135 degrees C . The column used was a 5% OV-17 column at an oven temperature of 135 C. The ordinate shows detector response . The abscissa shows retention
acid C. C. degrees time .
The NMR spectra of both methylated ISA and butanesulfonic acid showed the CH3 peak of the methylester as a singlet at 3 .88 ppm. A small singlet at 3 .30 ppm occurring in the spectrum of methylated ISA was assigned as the CH3 of the methylether of ISA on the basis of the known chemical shift value of 3 .38 ppm of the methylether singlet in methoxyethanol (10) . Integration of the spectrum indicated an approximate ratio of methylester CH3 to methylether CH3 of 20 :1 .
Isethionic Acid In Mammalian Tissues
Vol . 20, No . 12, 1977
100
2033
Spectrum A
5e
C
N SOCN~ .CIL~
J
ô
MMhyl.stor mothylofher of Istrthionic Acid
28 31
m > â Ca,o~ . m
CcwFNjOcq f
-1
m/e Spectrum B
29
j
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27
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.
CN70N
J*
g1.CN01~
C O
79
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o=
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Mothylnstor of Is .fhionic Acid
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96
.
65 t
97
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]. SOCIH3
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yl~
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`i. .
m/e FIG . 2 Mass spectra of the products of methylation of isethionic acid . A. Mass spectrum of the Peak I from Figure 1 . B. Mass spectrum of Peak II from Figure 1 . Experimental conditions and interpretation of data are described in the text .
Figure 3 shows a typical standard curve . When the method was used to analyse ISA in biological samples, a small peak in the position of ISA was detected in rat brain at a concentration of approximately 0.2 mg/l00g of tissue and in rat heart at a concentration of approximately 0 .10 mg/1008 tissue . This value for rat heart is only an estimate because at this level we are at the approximate limit of sensitivity for the assay technique. We were unable to detect any ISA in dog heart . The analytical procedure was always monitored by adding ISA at concentrations of 2 .0 and 0 .2 umole/g of tissue to duplicate aliquots of tissue examined . Recovery was always between 95 and 100% . The method as described using flame ionization detection is capable of detecting ISA in tissue as a concentration of approximately 0 .2 umole ISA/gram of tissue extracted (approximately 2 .5 mg/1008) . With the sulfur detector the sensitivity of the method was approximately 0 .008 umoles/gm (0 .1 mg/l00g tissue) . In squid axoplasm, isethionic acid was found at a concentration of 150 umole per ml axoplasm . The identity of the peaks was confirmed by gas chromatography mass-spectrometry.
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Isethionic Acid In Mammalian Tissues
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9.0
8.0 7.0 6.0 U Q
a
5 .0
C V 4.0 O u
Wr V) II
O .C oc
3.0
2.0
0.5
1 .0
1.5
2 .0
2 .5
3.0
-ttmole Isethionic Acid/Vial
FIG . 3 Calibration curves of methylated isethionic acid obtained on a column of 5% OV-17 on H.P . Chromosorb W . Operating parameters are quoted in the text . DISCUSSION Methylation of ISA produces two compounds, a methylester and the doubly methylated methylester, methylether derivative . These two compounds co-elute when analyzed by gas-liquid chromatography on a column of OV-1, but can be separated on a column of OV-17 (see Fig. lß) . The ratio of these two compounds separated on OV-17 is approximately 20 :1 with the methylester derivative being predominant . The ratio of the two peaks was invariant over a wide range of gas chromatographic conditions . The ratio was confirmed by proton NMR spectroscopy . The assignment of structures to the mass spectra of the peaks by methylation of ISA (Fig . 2) follows the discussion of fragmentation ~atterns of alkyl alkanesulfonates by Truce et al . (11) . The parent ions {M) . were not seen in the methylated ISA mass spectra. Truce et al . claim that these ions are scarce for most of the alkyl alkanesulfonates . However, the assignment of structures of the two peaks were strengthened by the appearance of the {M-1}t fragment for the methylester of ISA at m/e - 139 . We found that it was usually necessary to use two columns, one of OV-1 and the other of OV-17, and occasionally two methods of detection, flame ionization and flame photometric (for sulfur) to look at analytical extracts of tissues . This was necessary because all of the tissues studied gave a peak in the position of
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Isethionic Acid In Mammalian Tissues
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ISA in the crude extracts of rat and dog heart and rat brain studied when an OV-1 column was used . The peaks on OV-1 chromatography corresponded to an amount of material that would have been approximately 10-12 mg ISA/1008 heart or brain if they had been ISA. Confirmation that this peak was not ISA depended on rechromatography of the same extract on OV-17, and verification that this peak did not contain sulfur . The large peak from heart and brain extracts seen on OV-1 proved to contain only very small amounts of ISA when reassessed using OV-17 and the sulfur detector . The value that we obtained for the analysis of ISA in the squid giant axon compares favorably with other data obtained using different, less sensitive methods where 150 umole ISA per g . axoplasm has been reported (12,13) . Welty et al . (3) quoted a figure of 42 .6 mg ISA per 100g rat heart tissue and 12 .9 mg per 1008 dog heart. These amounts would have been quite easily detectable with our method . Using the sulfur detector which, in this particular case, was ten times more sensitive than the flame ionization detector, only a very small peak in the position of ISA could be seen for rat heart at a concentration of roughly 0 .10 mg/1008. Insufficient material was available to confirm its identity by mass spectrometry . In the case of rat brain, a peak could be seen with the sulfur detector at the position of ISA, at a concentration of 0 .2 mg/ 100g of tissue . Again, insufficient material was available to confirm that this small amount of material was truly ISA . We extracted as much as 400g of dog heart tissue to search for ISA. In this large scale experiment we also found In this large scale experiment we followed exactly the procedure no ISA peak . of Welty, Read and Shaw (3) . We obtained neither the crystals of sodium isethionate that they reported nor gas chromatographic evidence of ISA . We can not explain the difference between our results and those of Welty, Read and Shaw (3) . The sensitivity for the gas-liquid chromatographic portion of this experiment would have been approximately 0.1 mg/l00g of heart tissue . Our findings cast doubt on theories of the mode of action of taurine which involve bioconversion of taurine to ISA . This work was presented at the 2nd International Congress on Taurine in Tucson, Arizona, March 1977, and a portion of it has appeared in abstract form (14) . ACKNOWLEDGEMENTS We wish to thank the B .C . Heart Foundation for a grant-in-aid . We also wish to thank Mr . Greg Owen, Department of Chemistry, Simon Fraser University for help in obtaining mass spectra. REFERENCES J . G. JACOBSEN and L . H. SMITH, JR, Physiol. Rev . 48 424-511 (1968) . B . A. KOECHLIN, J . Biophys and Biochem . Cytol. _ 1 511-529 (1955) . J . D. WELTY, W. 0. READ and E . H . SHAW, J . Bioi . Chem . 237 1150-1161 (1962) . W. 0. READ and J .D . WELTY, J . Biol . Chem . 237 1521-1522 (1962) . E . J . PECK, JR, and J . AWAPARA, Biochem. Biophys . Acts 141 499-506 (1967) . E . I . CHAZOV, L . S . MALCHIKOVA, N . V . LIPINA, G . B . ASAFOV and V. N. SMIRNOV, Circ . Res . 34-35 III-11 (1974) . 7. W. 0 . READ and J .D . WELTY, Electrolyte and Cardiovascular Diseases, E . BAJUSZ, Ed . pp . 70-85, S . KARGER, Basel New York (1965) . 8. J . G. JACOBSEN, L. L . COLLINS and L . H. SMITH, JR, Nature 214 1247-1248 (1967) . 9. I . LEHTINEN and R. S . PIRA, Comm . 9th Inter. Congr. of Biochem . p . 446 (1973) . 10 . THE SADTLER STANDARD NUCLEAR MAGNETIC RESONANCE SPECTRUM , #32M . Sadtler Research Inc ., Pub . by Sadtler Research Laboratories, 3316 Spring Gardens 1. 2. 3. 4. 5. 6.
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Street, Philadelphia PA . 19104, USA (1967) . 11 . W . E . TRUCE, E . W . CAMPBELL and G . D . MADDING, J. Org . Chem . 32 308-317 (1967) . 12 . G . G . J . DEFFNER and R. E. RAFTER, Biochem. Biophgs . Acta 42 200-205 (1960) . 13 . F. C . G . HOSKIN and M . BRANDS, J . Neurochem. _20 1317-1327 (1973) 14 . D. A . APPLEGARTH, M. REMTULLA and I . R. WILLIAMS, Clin . Res . XXIV, 646A (1976) .
Pergamon Press
ANALYSIS OF ISETHIONIC ACID IN MAMMALIAN TISSUES Mohamed A . Remtulla, Derek A . Applegarth, Donald G . Clark and Ian H . Williams Departments of Paediatrics, Pathology and Chemistry, The University of British Columbia, Biochemical Diseases Laboratory, Children's Hospital and Government of Canada Agriculture Research Station, Vancouver, British Columbia (Received in final form May 17, 1977) SUMMARY
A gas-liquid chromatographic assay has been developed to measure isethionic acid after methylation with diazomethane . The identity of the products o£ methylation has been confirmed by mass-spectrometry and nuclear magnetic resonance spectroscopy . The method was used to measure isethionic acid in rat heart, dog heart and rat brain . The assay was validated by measuring isethionic acid on squid axoplasm . We have been able to detect only trace amounts of isethionic acid in rat brain (0 .2 mg/1008) and rat heart (0 .1 mg/ 1008) . None was found in dog heart . Isethionic acid, 2-hydroxy-ethane sulfonic acid, is the deaminated analogue of Its presence in biological material was first reported by taurine (1) . Roechlin (2) who found that it was the major anion of the axoplasm from the squid giant axon . He suggested that ISA might indirectly be responsible for the production of electrical phenomena in the nerve . Welty et al . (3) proposed that taurine was the precursor of ISA. These workers apparently isolated substantial quantities of ISA from dog and rat heart tissues, by a gravimetric method involving the crystallization of ISA as its sodium salt from a hot aqueous extract of dog heart or rat heart . They later demonstrated the conversion of 35 S-taurine to 35 S-ISA by dog heart slices (4) . Later Peck and Awapara (5) reported that very small amounts of isethionic acid could be formed from isotopically labelled taurine in rat heart and brain tissues . Other workers (6,7) have suggested that the conversion of taurine to ISA in myocardial cells facilitates the retention of intracellular calcium or potassium ions . The major difficulty of studying the function of ISA has been the lack of a good analytical procedure for its detection and quantitation . Methods used in the past to detect ISA do not offer much accuracy or sensitivity and some apparently promising methods have never been published in full (8,9) . We therefore report here an analytical method to measure ISA. MATERIALS AND METHODS For analysis of rat tissues, we used Wistar rats weighing approximately 200g . Animals were sacrificed by a sharp blow to the head . Hearts and brains were promptly excised and the tissues rinsed in normal saline, blotted on Wha,man Correspondence to : Derek A. Applegarth, Biochemical Diseases Laboratory, Children's Hospital, 250 West 59th Avenue, Vancouver, B.C . Canada V5X 1X2 . 2029
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#1 filter paper, and immediately frozen in small plastic vials in liquid nitrogen . This process required less than ten minutes per rat . For analysis of dog heart, animals were obtained from the Department of Physiology at The University of British Columbia, after having been used for open heart surgery . The dogs were sacrificed with 15% potassium chloride (10 ml) and the heart stored at -20 degrees before use . Samples of the giant axon of squid (Loligo pealii) were obtained from Dr . F .C .G . Hoskin of the Department of Biology, Illinois Institute of Technology, Chicago, Illinois . Isolation of ISA from Heart and Brain Tissues : 5g samples of pooled brain or heart tissues were used for experimentation . The heart tissue was minced before being used and then divided into two equal portions of 2 .5g each . To one portion was added 1 .0 ymole of sodium isethionate (Sigma Chemical Company, St . Louis, Missouri) . The other portion was used without any addition . Both portions were homogenized in 50% methanol/water (v/v), (10 ml), in a Sorvall omnimixer (Ivan Sorvall Inc ., Norwalk, Connecticut), using a teflon chamber at 3/4 of the full speed for five minutes, followed by one minute of full speed. The homogenate was transferred to a centrifuge tube and centrifuged at 3,000g for five minutes . The homogenizing chamber was rinsed three times with 5 ml, 50% methanol/water (v/v), and the rinse washings added to the centrifuge pellet which was suspended in the rinsing solution using a Vortex mixer . The resulting suspension was then centrifuged again for five minutes and the supernatant fluid removed . To the combined volume of supernatants and rinsings, an equal volume of Folch solvent (chloroform:methanol, 2:1, v/v) was added . The solutions were mixed thoroughly and centrifuged to separate the layers . The upper aqueous layer was removed and evaporated to dryness in a rotary evaporator under reduced pressure . After evaporation of the aqueous layer to dryness, 2 ml of a purified cation exchange resin (AG-50W-X8, 50-100 mesh, H+ form Bio Rad Laboratories, Richmond, California), prewashed in methanol and suspended in an equal volume of methanol, was added to the flask. After trituration, the mixture was transferred to a 5 ml glass-stoppered conical centrifuge tube . After centrifugation, the methanol layer was dried in a vacuum desiccator over sulphuric acid . Isolation of ISA from Axoplasm of the Squid Giant Axon : Axoplasm from the squid giant axon was obtained in freeze-dried form . The sample, 83mg fresh weight axoplasm, was dispersed in deionized water using a glass homogenizer . The turbid solution so formed was made up to a volume of 10 ml with deionized water . An aliquot of the solution was mixed with an equal volume of absolute methanol and this mixture treated with an equal volume of Folch solvent . The rest of the procedure was the same as that described above, for isolation of ISA from heart and brain tissues . Preparation of Standards : A suspension of ion exchange resin AG-50 (H+ form in methanol, 1 ml of resin + 1 ml of methanol) was pipetted into each of six, 5 ml glass stoppered, calibrated, conical centrifuge tubes . After allowing the resin to settle, the methanol layer was aspirated and discarded . To each tube was added 0, 0.1, 0 .2, 0.3, 0 .4, 0.5 and 0 .6 ml aliquots of 5 mM sodium isethionate solution in water. Volume was made up to 4 ml in each tube with methanol, and the resin suspended using a Vortex mixer. After centrifugation, the methanol layer from each tube was removed into reaction vials and evaporated to dryness in a vacuum desiccator over sulphuric acid ._ Abbreviations : Throughout the text, isethionic acid is abbreviated as ISA. Nuclear magnetic resonance spectroscopy is abbreviated as NMR .
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Isethionic Acid In Mammalian Tissues
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Methylation of the Samples and Preparation of a Standard Curve : ISA standards, or samples obtained from a tissue extract, were dissolved in 0 .1 ml of a 1 MM solution of salicylic acid (Sigma Chemical Company, St . Louis, Missouri) in methanol . Salicylic acid was used as an internal standard . The vials were stoppered using teflon laminated discs and placed in an ice-bath for five minutes . Ethereal diazomethane solution prepared from diazald (Aldrich Chemical Company, Milwaukee, Wisconsin) was introduced into the vials slowly, with mixing until the yellow color persisted . An additional three drops of diazomethane solution was added and the mixture was allowed to stand for about thirty minutes The total volume in the vial was 0.2 ml or less . Where the total in ice . volume in the vial was more than 0.2 ml, the excess solvent was carefully evaporated under a stream of dry nitrogen and the sample remethylated for a further thirty minute period . Methylation reactions were all stopped by adding one drop 50% acetic acid in water (v/v) . This remethylation procedure was found not to affect the standard calibration curve for ISA . The gas-liquid chromatoGas-Liquid Chromatography : Flame Ionization Detection : graph used was a Bendix,Model 2500, equipped with a flame ionization detector . Columns were 6 ft by 4 mm (i .d .) glass U-tubes . The stationary phases used were 5% OV-1 and 5% OV-17 (Gas Chromatographic Specialities Ltd . ; Brockville, Ontario) on 80-100 mesh H.P . Chromosorb W. Analyses were performed isothermally at temperatures ranging from 100 degrees C to 150 degrees C . The optimal temperature for OV-1 was 115 degrees C and for OV-17 was 135 degrees C . Nitrogen was used as a carrier gas at a flow rate of 40 ml/min . Gas-Liquid Chromatography : Sulphur Detection : The gas-liquid chromatograph used for such experiments was a Micro Tek 220 equipped with a flame photometric detector, Model FPD 100 (Melpar Inc ., Falls Church, Virginia) . The column used was a~6 ft by 2 mm (i .d .) glass U-tube with 5% OV-17 on 80-100 mesh H.P . Chromosorb W . Nitrogen was used as the carrier gas with an inlet flow of 30 ml/min . Column temperature was 100 degrees C . Oxygen flow to the detector was 10 ml/min hydrogen flow was 70 ml/min and air flow was 30 ml/min . Gas-Liquid Chromatography : Mass Spectrometry : The mass-spectrometer used was a Hitachi Perkin-Elmer, operating at an ionization energy of 70 ev . interfaced with a Varian, Model 1400 gas chromatograph . Authentic ISA and butane sulfonic acid samples were analyzed on OV-17 column after methylation with diazomethane . Chromatographic details were as outlined above for flame ionization detection . Nuclear Magnetic Resonance Spectroscopy : For proton NMR spectroscopy, the methyl esters of ISA and butane sulfonic acid were prepared in the following manner . Five times recrystallized sodium isethionate (100 mg) in boiling absolute ethanol and 1-butanesulfonic acid sodium salt (100 mg) (Eastman Kodak Company, Rochester, N.Y .) were each dissolved in methanol (1 .0 ml) and the samples treated with resin, and methylated as described above . After thirty minutes, excess diazomethane was carefully blown off with a stream of dry nitrogen and the sample completely dried down under reduced pressure over sulphuric acid . The NMR spectra of the reaction products, without further purification, were taken at 100 mHz in a Varian HA-100 spectrometer . Samples were dissolved in deuterated dimethylsulfoxide (Merck, Sharpe and Dohme Ltd., Canada) with tetramethyl silane being used as an internal standard . For the purposes of comparison, the NMR spectrum of crystallized, non-methylated ISA, also dissolved in dimethylsulfoxide was obtained . RESULTS Chromatography of the methylated ISA on OV-1 and on an OV-17 column is shown in Figure 1. Using a column of 5% OV-1, a single peak with a retention time of 1 .6 minutes was obtained for methylated ISA . Using a column of 5% OV-17, two
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peaks at retention times of 3 .5 and 4 .0 minutes were obtained . The identity of these two peaks was established by the use of gas chromatography mass-spectrometry (Figure 2) and nuclear magnetic resonance spectroscopy . On OV-17 the peak area of the large peak was approximately 20 times that of the smaller peak . Interpretation of the mass-spectrometry fragmentation pattern of the two peaks obtained after OV-17 gas chromatography is shown in Figure 2 . The large peak (Peak II) was the methylester of the ISA, while the small peak (Peak I) was the methylether, methylester of ISA.
B
A
~Solvent Peak
C
f- Solvent Peak
"--- Peak
Peak II 11
Isethionic Acid
Salicylic Acid Peak
1
Salicylic Acid
FIG . 1 Chromatographic separation of the products of methylation of isethionic and salicylic acid using flame ionization detection . A. The column used was a 5% OV-1 column ; oven temperature 115 degrees B. The column used was a 5%OV-17 column ; oven temperature 135 degrees C . The column used was a 5% OV-17 column at an oven temperature of 135 C. The ordinate shows detector response . The abscissa shows retention
acid C. C. degrees time .
The NMR spectra of both methylated ISA and butanesulfonic acid showed the CH3 peak of the methylester as a singlet at 3 .88 ppm. A small singlet at 3 .30 ppm occurring in the spectrum of methylated ISA was assigned as the CH3 of the methylether of ISA on the basis of the known chemical shift value of 3 .38 ppm of the methylether singlet in methoxyethanol (10) . Integration of the spectrum indicated an approximate ratio of methylester CH3 to methylether CH3 of 20 :1 .
Isethionic Acid In Mammalian Tissues
Vol . 20, No . 12, 1977
100
2033
Spectrum A
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.
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m/e FIG . 2 Mass spectra of the products of methylation of isethionic acid . A. Mass spectrum of the Peak I from Figure 1 . B. Mass spectrum of Peak II from Figure 1 . Experimental conditions and interpretation of data are described in the text .
Figure 3 shows a typical standard curve . When the method was used to analyse ISA in biological samples, a small peak in the position of ISA was detected in rat brain at a concentration of approximately 0.2 mg/l00g of tissue and in rat heart at a concentration of approximately 0 .10 mg/1008 tissue . This value for rat heart is only an estimate because at this level we are at the approximate limit of sensitivity for the assay technique. We were unable to detect any ISA in dog heart . The analytical procedure was always monitored by adding ISA at concentrations of 2 .0 and 0 .2 umole/g of tissue to duplicate aliquots of tissue examined . Recovery was always between 95 and 100% . The method as described using flame ionization detection is capable of detecting ISA in tissue as a concentration of approximately 0 .2 umole ISA/gram of tissue extracted (approximately 2 .5 mg/1008) . With the sulfur detector the sensitivity of the method was approximately 0 .008 umoles/gm (0 .1 mg/l00g tissue) . In squid axoplasm, isethionic acid was found at a concentration of 150 umole per ml axoplasm . The identity of the peaks was confirmed by gas chromatography mass-spectrometry.
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Isethionic Acid In Mammalian Tissues
Vol. 20, No . 12, 1977
9.0
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FIG . 3 Calibration curves of methylated isethionic acid obtained on a column of 5% OV-17 on H.P . Chromosorb W . Operating parameters are quoted in the text . DISCUSSION Methylation of ISA produces two compounds, a methylester and the doubly methylated methylester, methylether derivative . These two compounds co-elute when analyzed by gas-liquid chromatography on a column of OV-1, but can be separated on a column of OV-17 (see Fig. lß) . The ratio of these two compounds separated on OV-17 is approximately 20 :1 with the methylester derivative being predominant . The ratio of the two peaks was invariant over a wide range of gas chromatographic conditions . The ratio was confirmed by proton NMR spectroscopy . The assignment of structures to the mass spectra of the peaks by methylation of ISA (Fig . 2) follows the discussion of fragmentation ~atterns of alkyl alkanesulfonates by Truce et al . (11) . The parent ions {M) . were not seen in the methylated ISA mass spectra. Truce et al . claim that these ions are scarce for most of the alkyl alkanesulfonates . However, the assignment of structures of the two peaks were strengthened by the appearance of the {M-1}t fragment for the methylester of ISA at m/e - 139 . We found that it was usually necessary to use two columns, one of OV-1 and the other of OV-17, and occasionally two methods of detection, flame ionization and flame photometric (for sulfur) to look at analytical extracts of tissues . This was necessary because all of the tissues studied gave a peak in the position of
Vol. 20, No . 12, 1977
Isethionic Acid In Mammalian Tissues
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ISA in the crude extracts of rat and dog heart and rat brain studied when an OV-1 column was used . The peaks on OV-1 chromatography corresponded to an amount of material that would have been approximately 10-12 mg ISA/1008 heart or brain if they had been ISA. Confirmation that this peak was not ISA depended on rechromatography of the same extract on OV-17, and verification that this peak did not contain sulfur . The large peak from heart and brain extracts seen on OV-1 proved to contain only very small amounts of ISA when reassessed using OV-17 and the sulfur detector . The value that we obtained for the analysis of ISA in the squid giant axon compares favorably with other data obtained using different, less sensitive methods where 150 umole ISA per g . axoplasm has been reported (12,13) . Welty et al . (3) quoted a figure of 42 .6 mg ISA per 100g rat heart tissue and 12 .9 mg per 1008 dog heart. These amounts would have been quite easily detectable with our method . Using the sulfur detector which, in this particular case, was ten times more sensitive than the flame ionization detector, only a very small peak in the position of ISA could be seen for rat heart at a concentration of roughly 0 .10 mg/1008. Insufficient material was available to confirm its identity by mass spectrometry . In the case of rat brain, a peak could be seen with the sulfur detector at the position of ISA, at a concentration of 0 .2 mg/ 100g of tissue . Again, insufficient material was available to confirm that this small amount of material was truly ISA . We extracted as much as 400g of dog heart tissue to search for ISA. In this large scale experiment we also found In this large scale experiment we followed exactly the procedure no ISA peak . of Welty, Read and Shaw (3) . We obtained neither the crystals of sodium isethionate that they reported nor gas chromatographic evidence of ISA . We can not explain the difference between our results and those of Welty, Read and Shaw (3) . The sensitivity for the gas-liquid chromatographic portion of this experiment would have been approximately 0.1 mg/l00g of heart tissue . Our findings cast doubt on theories of the mode of action of taurine which involve bioconversion of taurine to ISA . This work was presented at the 2nd International Congress on Taurine in Tucson, Arizona, March 1977, and a portion of it has appeared in abstract form (14) . ACKNOWLEDGEMENTS We wish to thank the B .C . Heart Foundation for a grant-in-aid . We also wish to thank Mr . Greg Owen, Department of Chemistry, Simon Fraser University for help in obtaining mass spectra. REFERENCES J . G. JACOBSEN and L . H. SMITH, JR, Physiol. Rev . 48 424-511 (1968) . B . A. KOECHLIN, J . Biophys and Biochem . Cytol. _ 1 511-529 (1955) . J . D. WELTY, W. 0. READ and E . H . SHAW, J . Bioi . Chem . 237 1150-1161 (1962) . W. 0. READ and J .D . WELTY, J . Biol . Chem . 237 1521-1522 (1962) . E . J . PECK, JR, and J . AWAPARA, Biochem. Biophys . Acts 141 499-506 (1967) . E . I . CHAZOV, L . S . MALCHIKOVA, N . V . LIPINA, G . B . ASAFOV and V. N. SMIRNOV, Circ . Res . 34-35 III-11 (1974) . 7. W. 0 . READ and J .D . WELTY, Electrolyte and Cardiovascular Diseases, E . BAJUSZ, Ed . pp . 70-85, S . KARGER, Basel New York (1965) . 8. J . G. JACOBSEN, L. L . COLLINS and L . H. SMITH, JR, Nature 214 1247-1248 (1967) . 9. I . LEHTINEN and R. S . PIRA, Comm . 9th Inter. Congr. of Biochem . p . 446 (1973) . 10 . THE SADTLER STANDARD NUCLEAR MAGNETIC RESONANCE SPECTRUM , #32M . Sadtler Research Inc ., Pub . by Sadtler Research Laboratories, 3316 Spring Gardens 1. 2. 3. 4. 5. 6.
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Street, Philadelphia PA . 19104, USA (1967) . 11 . W . E . TRUCE, E . W . CAMPBELL and G . D . MADDING, J. Org . Chem . 32 308-317 (1967) . 12 . G . G . J . DEFFNER and R. E. RAFTER, Biochem. Biophgs . Acta 42 200-205 (1960) . 13 . F. C . G . HOSKIN and M . BRANDS, J . Neurochem. _20 1317-1327 (1973) 14 . D. A . APPLEGARTH, M. REMTULLA and I . R. WILLIAMS, Clin . Res . XXIV, 646A (1976) .