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Chemical methods for the determination of acetylcholine
Life Sciences, Vol. 58, No. 22, pp. 1917-1919. 19% Gqyright 0 1996 Elsevia Science Inc. Printed in the USA. All rights rtscrvcd 0024-3205/% $15.00 t .OLl
ELSEVIER
PI1 SOO24-3205(96)00180-4
CHEMICAL METHODS FOR THE DETERMIN ATION OF ACETYLCHOLINE Bo Holmstedt Department of Toxicology Karolinska Institutet Box 210 S-171 77 Stockholm Sweden
I met Donald Jenden for the first time in the summer of 1953 at UCLA. Since then, we have been in close contact and sometimes have worked together. Especially memorable is the time that Don and his co-worker, Israel Hanin, spent in my laboratory during 1968, when we worked on the determination of acetylcholine (ACh) in fresh brain tissue. Although Stedman and Stedman in 1937 were able to recover ACh chemically as synthesized in a brain homogenate (l), it was particularly the development of gas chromatography-mass spectrometry that allowed the determination of ACh in submicrogram quantities of fresh brain substance. We succeeded in proving by physical-chemical methods the occurrence of acetylcholine in fresh rat brain, at the same time excluding propionylcholine and butyrylcholine (2). This prompted the letter shown as Insert 1 to Sir Henry Dale, then living in retirement in Cambridge. The reply from Sir Henry Dale is reproduced as Insert 2. One must forgive the aged scientist for not giving the correct reference to the discovery of propionylcholine in ox spleen. This was discovered by Banister and her co-investigators (3,4). Recent confirmation is available. The letter shown in Insert 2 must have been the last thing Dale ever wrote about ACh. Two weeks afterward he had passed away. Stockholm,June 7, 1968 Dear Sir Henry: It is nowforty years since you and Dud& reported your classical work in the first rigorous
i&nt@ation of acetylcholinein animal tissues. Althoughmost of us have relied on a large volume of ci~umstantial evidence for its occurrence in mammalian brain, no positive id&ijkation in fresh bmin has so far been reported. We themfoorebelieve it may be of interestto you to know thatfollow@ the developmentof a gas chmmatogmphic technique for microanalysisof choline esters at the Depamnent of Pharmacology, Uniwxvity of califrnia, Los Angeles and the combination of gas chromatographyand mass spectmmetrywhich has been pioneered at the Kamlinska Institute
1918
Determination of Acetylcholine
Vol. 58, No. 22, 1996
in Stockholm, we succeeded on the foutth of this month in conclusively aimwnstmting the existence and i&r&y of acetylcholine in a single fresh rat brain. Our pxperiments have also &monstrated that propionylcholine and butyrylcholine do not occur in this tissue in significant amounts (less that O.&I1 j&g). In view of the tremendous technological advances in recent years w cannot but feel humble at your great achievements with such simple means forty years ago. Yours respectively, Bo Hobnstedt Professor Dept. of Toxicology Karolinska Instituter Stockholm
Donatd J. Jet&n Professor Dept. of Pharmacology University of California Los Angeles
Insert 1
14th June, 1968 Dear Professors Hobnstedt and Jenden: Your very kind, and extremely interesting letter of June 7th, was duly forwarded to me at this, which is now my permanent, address in Cambridge, where 1 have been obliged to retire on account of a series of acci&nts, which I had the misfortune to suffer now three years ago, on, and @et, what was then my %kh birthday. You will realize thus, perhaps, that your letter came to me as a specially cheering item of a biggish consignment of letters, greeting cards and cables, which I was so happy as to receive here,from various parts of the n&d, in celebration of my 93td birthday, on Sun&v, June 9rh, now nearly a week ago. I do congratulate you very heartily on your success in &monstrating the presence of acetylcholine in a brain, which must have been almost living when you put it, and distributed it, in whateverjMtive medium you may have employed. You will agree that one of the interesting and puzzling questions still to be answered, is the apparently random distribution of acetylcholine in the organs of di$erent species, from which it can be easily obtained by metW far less subtle and conservative, than those which you have now been able to appEy.As you are aware, my late colkague, Harold Dud@ and I came across it abnost by accident, in the spleens of the large ungulates, and actually managed to isolate it and i&n&B it fromthe spleen of the horse. Later, as you are a2x&tless aware, Dr. Catherine Hebb, of the Agricultural Research Station near here, had the curiosity to isolate, from the spleen of the ox, what Dudky and I had naturally thought also to be acetylcholine, but which she now found to be a mixture of acetyl and propionyl cholines. Still more surprising, was the discovery by my colleague, J.H. Gaddum, while I war still in action, that the human placenta contains a higher proportion of acet#toline than any other organ of any species, which we examined at that time. But nobody, even yet, has been able to guess what it is doing there, and why, of the animal placentae which have yet been examined in that connexion, the human placenta is still the onty one in which it has been discovered; there in such an extraordinary amount, but with no nerwus tissue to accountjiw its presence.
Vol. 58, No. 22, 1996
Determinationof Acetylcholine
1919
I do again send you my heartiest thanksfor your most intetwing communicationand your kindly refwnce to whatwere, necessatity,in those earlier days, the relativelyen& methoa!v which my colleagues and I were then obliged to employ. Yours sincerely, Henry H. Dale
1. 2. 3. 4.
Insert 2
E. STEDMAN and E. STEDMAN, Biachem. J. 2 817-827 (1937). C.G. HAMMAR, I. HANIN, B. HOLMSTEDT, R. KIT& D.J. JENDEN and B. KARLI%, Nature a 915-917 (1968). R.J. BANISTER, V.P. WHIlTAKER and S. WIJESUNDERA, J. Physiol. (Land.), (Proc. Physiol. Sot.) m 55P (1951). R. J. BANISTER, V.P. WHITTAKER and S. WIJESUNDERA, J. Physiol. (Land) 121 55-71 (1953).
ELSEVIER
PI1 SOO24-3205(96)00180-4
CHEMICAL METHODS FOR THE DETERMIN ATION OF ACETYLCHOLINE Bo Holmstedt Department of Toxicology Karolinska Institutet Box 210 S-171 77 Stockholm Sweden
I met Donald Jenden for the first time in the summer of 1953 at UCLA. Since then, we have been in close contact and sometimes have worked together. Especially memorable is the time that Don and his co-worker, Israel Hanin, spent in my laboratory during 1968, when we worked on the determination of acetylcholine (ACh) in fresh brain tissue. Although Stedman and Stedman in 1937 were able to recover ACh chemically as synthesized in a brain homogenate (l), it was particularly the development of gas chromatography-mass spectrometry that allowed the determination of ACh in submicrogram quantities of fresh brain substance. We succeeded in proving by physical-chemical methods the occurrence of acetylcholine in fresh rat brain, at the same time excluding propionylcholine and butyrylcholine (2). This prompted the letter shown as Insert 1 to Sir Henry Dale, then living in retirement in Cambridge. The reply from Sir Henry Dale is reproduced as Insert 2. One must forgive the aged scientist for not giving the correct reference to the discovery of propionylcholine in ox spleen. This was discovered by Banister and her co-investigators (3,4). Recent confirmation is available. The letter shown in Insert 2 must have been the last thing Dale ever wrote about ACh. Two weeks afterward he had passed away. Stockholm,June 7, 1968 Dear Sir Henry: It is nowforty years since you and Dud& reported your classical work in the first rigorous
i&nt@ation of acetylcholinein animal tissues. Althoughmost of us have relied on a large volume of ci~umstantial evidence for its occurrence in mammalian brain, no positive id&ijkation in fresh bmin has so far been reported. We themfoorebelieve it may be of interestto you to know thatfollow@ the developmentof a gas chmmatogmphic technique for microanalysisof choline esters at the Depamnent of Pharmacology, Uniwxvity of califrnia, Los Angeles and the combination of gas chromatographyand mass spectmmetrywhich has been pioneered at the Kamlinska Institute
1918
Determination of Acetylcholine
Vol. 58, No. 22, 1996
in Stockholm, we succeeded on the foutth of this month in conclusively aimwnstmting the existence and i&r&y of acetylcholine in a single fresh rat brain. Our pxperiments have also &monstrated that propionylcholine and butyrylcholine do not occur in this tissue in significant amounts (less that O.&I1 j&g). In view of the tremendous technological advances in recent years w cannot but feel humble at your great achievements with such simple means forty years ago. Yours respectively, Bo Hobnstedt Professor Dept. of Toxicology Karolinska Instituter Stockholm
Donatd J. Jet&n Professor Dept. of Pharmacology University of California Los Angeles
Insert 1
14th June, 1968 Dear Professors Hobnstedt and Jenden: Your very kind, and extremely interesting letter of June 7th, was duly forwarded to me at this, which is now my permanent, address in Cambridge, where 1 have been obliged to retire on account of a series of acci&nts, which I had the misfortune to suffer now three years ago, on, and @et, what was then my %kh birthday. You will realize thus, perhaps, that your letter came to me as a specially cheering item of a biggish consignment of letters, greeting cards and cables, which I was so happy as to receive here,from various parts of the n&d, in celebration of my 93td birthday, on Sun&v, June 9rh, now nearly a week ago. I do congratulate you very heartily on your success in &monstrating the presence of acetylcholine in a brain, which must have been almost living when you put it, and distributed it, in whateverjMtive medium you may have employed. You will agree that one of the interesting and puzzling questions still to be answered, is the apparently random distribution of acetylcholine in the organs of di$erent species, from which it can be easily obtained by metW far less subtle and conservative, than those which you have now been able to appEy.As you are aware, my late colkague, Harold Dud@ and I came across it abnost by accident, in the spleens of the large ungulates, and actually managed to isolate it and i&n&B it fromthe spleen of the horse. Later, as you are a2x&tless aware, Dr. Catherine Hebb, of the Agricultural Research Station near here, had the curiosity to isolate, from the spleen of the ox, what Dudky and I had naturally thought also to be acetylcholine, but which she now found to be a mixture of acetyl and propionyl cholines. Still more surprising, was the discovery by my colleague, J.H. Gaddum, while I war still in action, that the human placenta contains a higher proportion of acet#toline than any other organ of any species, which we examined at that time. But nobody, even yet, has been able to guess what it is doing there, and why, of the animal placentae which have yet been examined in that connexion, the human placenta is still the onty one in which it has been discovered; there in such an extraordinary amount, but with no nerwus tissue to accountjiw its presence.
Vol. 58, No. 22, 1996
Determinationof Acetylcholine
1919
I do again send you my heartiest thanksfor your most intetwing communicationand your kindly refwnce to whatwere, necessatity,in those earlier days, the relativelyen& methoa!v which my colleagues and I were then obliged to employ. Yours sincerely, Henry H. Dale
1. 2. 3. 4.
Insert 2
E. STEDMAN and E. STEDMAN, Biachem. J. 2 817-827 (1937). C.G. HAMMAR, I. HANIN, B. HOLMSTEDT, R. KIT& D.J. JENDEN and B. KARLI%, Nature a 915-917 (1968). R.J. BANISTER, V.P. WHIlTAKER and S. WIJESUNDERA, J. Physiol. (Land.), (Proc. Physiol. Sot.) m 55P (1951). R. J. BANISTER, V.P. WHITTAKER and S. WIJESUNDERA, J. Physiol. (Land) 121 55-71 (1953).