Chromatographic separation of reaction products from the choline acetyltransferase and carnitine acetyltransferase assay: Differential ChAT and CrAT activity in brain extracts from Alzheimer's disease versus controls

Jason A. Bailey, Debomoy K. Lahiri

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

Choline acetyltransferase (ChAT) catalyzes the reaction between choline and acetylcoenzyme A (AcCoA) to form acetylcholine (ACh) in nerve terminals. ACh metabolism has implications in numerous aspects of physiology and varied disease states, such as Alzheimer's disease. Therefore a specific, sensitive, and reliable method for detecting ChAT enzyme activity is of great utility in a number of situations. Using an existing radionuclide-based enzyme activity assay, we have observed detectable ChAT signals from non-cholinergic cells, suggesting a contaminant in the assay producing an artifactual signal. Previous reports have suggested that L-acetylcarnitine (LAC) contaminates many assays of ChAT activity, because of difficulties in separating LAC from ACh by organic extraction. To determine the source of this hypothesized artifact and to rectify the problem, we have developed a paper chromatography-based assay for the detection of acetylcholine and other contaminating reaction products of this assay, including LAC. Our first goal was to develop a simple and economical method for resolving and verifying the identities of various reaction products or contaminants that could be performed in most laboratories without specialized equipment. Our second goal was to apply this separation method in postmortem human brain tissue samples. Our assay successfully detected several contaminants, especially in assays using brain tissue, and allowed the separation of the intended ACh product from these contaminants. We further demonstrate that this assay can be used to measure carnitine acetyltransferase (CrAT) activity in the same samples, and assays comparing ChAT and CrAT show that CrAT is highly active in neuronal tissues and in neuronal cell cultures relative to ChAT. Thus, the simple chromatography-based assay we describe allows the measurement of specific reaction products separated from contaminants using commonly available and inexpensive materials. Further, we show that ChAT activity is significantly reduced in brain extracts from Alzheimer's disease compared to controls. Chromatographic separation of specific ChAT and CrAT products in human brain extracts A simple chromatographic method separates choline acetyltransferase (ChAT) and carnitine acetyltransferase (CrAT) assay products from contaminants. Specific ChAT assay product was reduced in Alzheimer's disease brain extracts, and CrAT activity was high in brain and neuron culture extracts. Contaminants of ChAT assays were identified and specific products isolated, confirming reduced ChAT activity in Alzheimer's disease.

Original languageEnglish (US)
Pages (from-to)672-680
Number of pages9
JournalJournal of Neurochemistry
Volume122
Issue number4
DOIs
StatePublished - Aug 1 2012

Fingerprint

Carnitine O-Acetyltransferase
Disease control
Choline O-Acetyltransferase
Reaction products
Assays
Brain
Alzheimer Disease
Impurities
Acetylcholine
Acetylcarnitine
Enzyme activity
Tissue
Chromatography
Paper Chromatography
Enzyme Assays
Choline
Physiology
Radioisotopes

Keywords

  • autopsied cases
  • cortex
  • human brain
  • neurchemical assay
  • neuropathologic
  • synapse

ASJC Scopus subject areas

  • Biochemistry
  • Cellular and Molecular Neuroscience

Cite this

@article{28a5f4182a5a446993fef0c24a892aa8,
title = "Chromatographic separation of reaction products from the choline acetyltransferase and carnitine acetyltransferase assay: Differential ChAT and CrAT activity in brain extracts from Alzheimer's disease versus controls",
abstract = "Choline acetyltransferase (ChAT) catalyzes the reaction between choline and acetylcoenzyme A (AcCoA) to form acetylcholine (ACh) in nerve terminals. ACh metabolism has implications in numerous aspects of physiology and varied disease states, such as Alzheimer's disease. Therefore a specific, sensitive, and reliable method for detecting ChAT enzyme activity is of great utility in a number of situations. Using an existing radionuclide-based enzyme activity assay, we have observed detectable ChAT signals from non-cholinergic cells, suggesting a contaminant in the assay producing an artifactual signal. Previous reports have suggested that L-acetylcarnitine (LAC) contaminates many assays of ChAT activity, because of difficulties in separating LAC from ACh by organic extraction. To determine the source of this hypothesized artifact and to rectify the problem, we have developed a paper chromatography-based assay for the detection of acetylcholine and other contaminating reaction products of this assay, including LAC. Our first goal was to develop a simple and economical method for resolving and verifying the identities of various reaction products or contaminants that could be performed in most laboratories without specialized equipment. Our second goal was to apply this separation method in postmortem human brain tissue samples. Our assay successfully detected several contaminants, especially in assays using brain tissue, and allowed the separation of the intended ACh product from these contaminants. We further demonstrate that this assay can be used to measure carnitine acetyltransferase (CrAT) activity in the same samples, and assays comparing ChAT and CrAT show that CrAT is highly active in neuronal tissues and in neuronal cell cultures relative to ChAT. Thus, the simple chromatography-based assay we describe allows the measurement of specific reaction products separated from contaminants using commonly available and inexpensive materials. Further, we show that ChAT activity is significantly reduced in brain extracts from Alzheimer's disease compared to controls. Chromatographic separation of specific ChAT and CrAT products in human brain extracts A simple chromatographic method separates choline acetyltransferase (ChAT) and carnitine acetyltransferase (CrAT) assay products from contaminants. Specific ChAT assay product was reduced in Alzheimer's disease brain extracts, and CrAT activity was high in brain and neuron culture extracts. Contaminants of ChAT assays were identified and specific products isolated, confirming reduced ChAT activity in Alzheimer's disease.",
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TY - JOUR

T1 - Chromatographic separation of reaction products from the choline acetyltransferase and carnitine acetyltransferase assay

T2 - Differential ChAT and CrAT activity in brain extracts from Alzheimer's disease versus controls

AU - Bailey, Jason A.

AU - Lahiri, Debomoy K.

PY - 2012/8/1

Y1 - 2012/8/1

N2 - Choline acetyltransferase (ChAT) catalyzes the reaction between choline and acetylcoenzyme A (AcCoA) to form acetylcholine (ACh) in nerve terminals. ACh metabolism has implications in numerous aspects of physiology and varied disease states, such as Alzheimer's disease. Therefore a specific, sensitive, and reliable method for detecting ChAT enzyme activity is of great utility in a number of situations. Using an existing radionuclide-based enzyme activity assay, we have observed detectable ChAT signals from non-cholinergic cells, suggesting a contaminant in the assay producing an artifactual signal. Previous reports have suggested that L-acetylcarnitine (LAC) contaminates many assays of ChAT activity, because of difficulties in separating LAC from ACh by organic extraction. To determine the source of this hypothesized artifact and to rectify the problem, we have developed a paper chromatography-based assay for the detection of acetylcholine and other contaminating reaction products of this assay, including LAC. Our first goal was to develop a simple and economical method for resolving and verifying the identities of various reaction products or contaminants that could be performed in most laboratories without specialized equipment. Our second goal was to apply this separation method in postmortem human brain tissue samples. Our assay successfully detected several contaminants, especially in assays using brain tissue, and allowed the separation of the intended ACh product from these contaminants. We further demonstrate that this assay can be used to measure carnitine acetyltransferase (CrAT) activity in the same samples, and assays comparing ChAT and CrAT show that CrAT is highly active in neuronal tissues and in neuronal cell cultures relative to ChAT. Thus, the simple chromatography-based assay we describe allows the measurement of specific reaction products separated from contaminants using commonly available and inexpensive materials. Further, we show that ChAT activity is significantly reduced in brain extracts from Alzheimer's disease compared to controls. Chromatographic separation of specific ChAT and CrAT products in human brain extracts A simple chromatographic method separates choline acetyltransferase (ChAT) and carnitine acetyltransferase (CrAT) assay products from contaminants. Specific ChAT assay product was reduced in Alzheimer's disease brain extracts, and CrAT activity was high in brain and neuron culture extracts. Contaminants of ChAT assays were identified and specific products isolated, confirming reduced ChAT activity in Alzheimer's disease.

AB - Choline acetyltransferase (ChAT) catalyzes the reaction between choline and acetylcoenzyme A (AcCoA) to form acetylcholine (ACh) in nerve terminals. ACh metabolism has implications in numerous aspects of physiology and varied disease states, such as Alzheimer's disease. Therefore a specific, sensitive, and reliable method for detecting ChAT enzyme activity is of great utility in a number of situations. Using an existing radionuclide-based enzyme activity assay, we have observed detectable ChAT signals from non-cholinergic cells, suggesting a contaminant in the assay producing an artifactual signal. Previous reports have suggested that L-acetylcarnitine (LAC) contaminates many assays of ChAT activity, because of difficulties in separating LAC from ACh by organic extraction. To determine the source of this hypothesized artifact and to rectify the problem, we have developed a paper chromatography-based assay for the detection of acetylcholine and other contaminating reaction products of this assay, including LAC. Our first goal was to develop a simple and economical method for resolving and verifying the identities of various reaction products or contaminants that could be performed in most laboratories without specialized equipment. Our second goal was to apply this separation method in postmortem human brain tissue samples. Our assay successfully detected several contaminants, especially in assays using brain tissue, and allowed the separation of the intended ACh product from these contaminants. We further demonstrate that this assay can be used to measure carnitine acetyltransferase (CrAT) activity in the same samples, and assays comparing ChAT and CrAT show that CrAT is highly active in neuronal tissues and in neuronal cell cultures relative to ChAT. Thus, the simple chromatography-based assay we describe allows the measurement of specific reaction products separated from contaminants using commonly available and inexpensive materials. Further, we show that ChAT activity is significantly reduced in brain extracts from Alzheimer's disease compared to controls. Chromatographic separation of specific ChAT and CrAT products in human brain extracts A simple chromatographic method separates choline acetyltransferase (ChAT) and carnitine acetyltransferase (CrAT) assay products from contaminants. Specific ChAT assay product was reduced in Alzheimer's disease brain extracts, and CrAT activity was high in brain and neuron culture extracts. Contaminants of ChAT assays were identified and specific products isolated, confirming reduced ChAT activity in Alzheimer's disease.

KW - autopsied cases

KW - cortex

KW - human brain

KW - neurchemical assay

KW - neuropathologic

KW - synapse

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