THE DISTRIBUTION OF ACETYL‐CoA IN SPECIFIC AREAS OF THE CNS OF THE RAT AS MEASURED BY A MODIFICATION OF A RADIO‐ENZYMATIC ASSAY FOR ACETYLCHOLINE AND CHOLINE

THE DISTRIBUTION OF ACETYL‐CoA IN SPECIFIC AREAS OF THE CNS OF THE RAT AS MEASURED BY A... Abstract— A rapid and sensitive enzymatic assay for measuring picomole quantities of acetyl‐CoA, acetylcholine (ACh), and choline from the same tissue extract has been developed. After ACh and choline were extracted into 15% 1 N formic acid/85% acetone, the pellet was further extracted with 5% trichloroacetic acid (TCA) to remove the remaining acetyl‐CoA. The two extraction solvents were pooled and lipids, organic solvents, and TCA were removed first by a heptane‐chloroform wash followed by an ether extraction. In the acetyl‐CoA assay, endogenous ACh and choline were removed by extractions with sodium tetraphenylboron in butenenitrile prior to the enzymatic reactions. The acetyl‐CoA remaining in the aqueous phase was then converted enzymatically to labelled ACh in the presence of (Me‐14C)choline using choline acetyltransferase. The unreacted labelled precursor was converted to choline phosphate by the enzyme choline kinase. The (14C)ACh formed from acetyl‐CoA was extracted into sodium tetraphenylboron in butenenitrile and a portion of the organic phase containing the (14C)ACh was counted in a scintillation counter. Acetylcholine and choline were assayed from the same tissue extracts by a modification of the procedure by SHEA & APRISON (1973). Acetyl‐CoA levels in rat whole brain when killed by the near‐freezing procedure were found to be 5.50 ± 0.2 nmol/g. The content of acetyl‐CoA was the same whether the rats were killed by the near‐freezing method or by total freezing in liquid nitrogen. The levels of acetyl‐CoA did not change with time after death when the tissue was maintained at a temperature of −10°C. In the same tissue extracts from rat whole brain killed by the near‐freezing method, the content of ACh was 20.6 ± 0.7 nmol/g and choline 58.2 ± 1.2 nmol/g. Although reproducible, the level reported for choline is high when assayed under this condition. The content of choline however after total freezing was found to be 25.2 ± 2.0 nmol/g. The sensitivity (d. p. m. of sample twice blank) is 10 pmol for the acetyl‐CoA assay and 25 pmol for the ACh and choline assays. The regional distribution of these three compounds in the brain of rats as well as the content of acetyl‐CoA in heart, liver and kidney are presented. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Neurochemistry Wiley

THE DISTRIBUTION OF ACETYL‐CoA IN SPECIFIC AREAS OF THE CNS OF THE RAT AS MEASURED BY A MODIFICATION OF A RADIO‐ENZYMATIC ASSAY FOR ACETYLCHOLINE AND CHOLINE

Journal of Neurochemistry, Volume 28 (1) – Jan 1, 1977

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Publisher
Wiley
Copyright
Copyright © 1977 Wiley Subscription Services, Inc., A Wiley Company
ISSN
0022-3042
eISSN
1471-4159
DOI
10.1111/j.1471-4159.1977.tb07707.x
Publisher site
See Article on Publisher Site

Abstract

Abstract— A rapid and sensitive enzymatic assay for measuring picomole quantities of acetyl‐CoA, acetylcholine (ACh), and choline from the same tissue extract has been developed. After ACh and choline were extracted into 15% 1 N formic acid/85% acetone, the pellet was further extracted with 5% trichloroacetic acid (TCA) to remove the remaining acetyl‐CoA. The two extraction solvents were pooled and lipids, organic solvents, and TCA were removed first by a heptane‐chloroform wash followed by an ether extraction. In the acetyl‐CoA assay, endogenous ACh and choline were removed by extractions with sodium tetraphenylboron in butenenitrile prior to the enzymatic reactions. The acetyl‐CoA remaining in the aqueous phase was then converted enzymatically to labelled ACh in the presence of (Me‐14C)choline using choline acetyltransferase. The unreacted labelled precursor was converted to choline phosphate by the enzyme choline kinase. The (14C)ACh formed from acetyl‐CoA was extracted into sodium tetraphenylboron in butenenitrile and a portion of the organic phase containing the (14C)ACh was counted in a scintillation counter. Acetylcholine and choline were assayed from the same tissue extracts by a modification of the procedure by SHEA & APRISON (1973). Acetyl‐CoA levels in rat whole brain when killed by the near‐freezing procedure were found to be 5.50 ± 0.2 nmol/g. The content of acetyl‐CoA was the same whether the rats were killed by the near‐freezing method or by total freezing in liquid nitrogen. The levels of acetyl‐CoA did not change with time after death when the tissue was maintained at a temperature of −10°C. In the same tissue extracts from rat whole brain killed by the near‐freezing method, the content of ACh was 20.6 ± 0.7 nmol/g and choline 58.2 ± 1.2 nmol/g. Although reproducible, the level reported for choline is high when assayed under this condition. The content of choline however after total freezing was found to be 25.2 ± 2.0 nmol/g. The sensitivity (d. p. m. of sample twice blank) is 10 pmol for the acetyl‐CoA assay and 25 pmol for the ACh and choline assays. The regional distribution of these three compounds in the brain of rats as well as the content of acetyl‐CoA in heart, liver and kidney are presented.

Journal

Journal of NeurochemistryWiley

Published: Jan 1, 1977

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