Anticholinesterase: Mechanisms and Clinical Uses

Cholinergic neurotransmission serves as an indispensable regulatory mechanism across both the central and peripheral nervous systems, orchestrating cognitive processes, autonomic balance, and somatic neuromuscular signaling. Chemical compounds capable of attenuating the enzymatic breakdown of acetylcholine—collectively known as anticholinesterases or cholinesterase inhibitors—constitute one of the most therapeutically versatile and toxicologically significant classes of pharmacological agents in modern biomedical science. By preserving the synaptic residency time of endogenous acetylcholine, these compounds exert profound physiological actions that range from symptomatic stabilization in neurodegenerative disease to fatal autonomic hyperactivity in acute chemical exposure.

Anticholinesterase

1. Concise Definition

An anticholinesterase (also termed a cholinesterase inhibitor) is a chemical substance, physiological agent, or xenobiotic that inhibits the functional activity of the enzyme cholinesterase, specifically acetylcholinesterase (AChE) and/or butyrylcholinesterase (BChE). By impeding the rapid enzymatic hydrolysis of the neurotransmitter acetylcholine into acetate and choline, anticholinesterases promote the accumulation and prolonged biological half-life of acetylcholine within the synaptic cleft, neuroeffector junctions, and somatic neuromuscular endplates.

Depending on their molecular geometry and binding kinetics, anticholinesterases operate through reversible, pseudo-irreversible, or irreversible mechanisms. Reversible and carbamate-based agents are central to clinical practice for treating neuromuscular junction disorders, neurodegenerative dementias, and reversing pharmacological paralysis in surgical settings. Conversely, irreversible organophosphorus compounds predominantly serve as agricultural pesticides or potent chemical warfare nerve agents, demonstrating the dual-use nature of this critical pharmacological class.

2. Etymology & Linguistic Origin

The term anticholinesterase is an etymological construct derived from classical Greek and scientific Latin roots. The prefix anti- originates from the Ancient Greek ἀντί (antí), signifying “against,” “opposed to,” or “in counteraction.” The root choline traces back to the Greek χολή (cholē), meaning “bile,” commemorating the initial isolation of choline from bovine bile by German chemist Adolph Strecker in 1862. The combining form stear originates from the Greek στέαρ (stéar), denoting tallow or solid fat, reflecting the ester linkage formed with acetic acid.

The suffix -ase is the standard biochemical nomenclature adopted from the French enzyme diastase (from the Greek διάστασις, meaning separation), established by Émile Duclaux to denote an enzyme. Thus, the hybrid descriptor designates any agent acting “against the enzyme that degrades the ester of choline.” The word gained widespread academic currency throughout the 1930s and 1940s following landmark biochemical investigations by Henry Hallett Dale and Otto Loewi into chemical neurotransmission.

3. Pronunciation & Grammatical Form

The standard phonetic transcription of anticholinesterase in International Phonetic Alphabet (IPA) format is /ˌæn.ti.koʊ.lɪnˈɛs.tə.reɪs/ in American English and /ˌæn.tiˌkɒl.ɪnˈɛs.tər.eɪz/ in Received Pronunciation British English. Grammatically, the term functions primarily as a singular countable noun (plural: anticholinesterases) and frequently serves an attributive or adjectival modifier function, as observed in phrases such as “anticholinesterase therapy,” “anticholinesterase poisoning,” or “anticholinesterase medication.” Synonymous formulations widely accepted in clinical pharmacology include “cholinesterase inhibitor” (ChEI) and “acetylcholinesterase inhibitor” (AChEI).

4. Detailed Conceptual Explanation

Under baseline homeostatic conditions, synaptic termination within the cholinergic nervous system requires exceptionally swift kinetics. Unlike catecholaminergic synapses that rely principally upon presynaptic reuptake transporters, the cholinergic synapse depends almost exclusively on rapid enzymatic destruction. The physiological enzyme acetylcholinesterase is one of the most catalytically efficient biological catalysts known, operating near the theoretical limits of diffusion control. A single molecule of acetylcholinesterase can hydrolyze up to 25,000 molecules of acetylcholine per second. This rapid clearance allows high-frequency transmission of discrete, unambiguous action potentials across the neuromuscular junction and interneuronal pathways.

Anticholinesterases interfere with this catalytic machinery by binding directly to the enzyme’s active site or associated regulatory domains. The active site of acetylcholinesterase is situated at the bottom of a deep, narrow gorge approximately 20 angstroms deep, known as the active-site gorge. Within this structure reside two primary functional subdomains: the esteratic site and the peripheral anionic site (PAS). The esteratic site contains a classical catalytic triad consisting of serine (Ser203), histidine (His447), and glutamate (Glu334). The peripheral anionic site, positioned at the entrance of the gorge, captures acetylcholine molecules and guides them through electrostatic attraction down to the catalytic machinery.

When an anticholinesterase occupies or modifies these domains, catalytic turnover ceases or slows down drastically. As a direct consequence, released acetylcholine cannot be catabolized and remains within the synaptic space. This causes prolonged activation and desensitization dynamics at two major subfamilies of cholinergic receptors:

• Muscarinic Acetylcholine Receptors (mAChRs): G-protein-coupled receptors (subtypes M1 through M5) located throughout smooth muscle, cardiac conducting tissue, exocrine glands, and the cerebral cortex. Excessive muscarinic stimulation produces bronchoconstriction, bradycardia, miosis, hyperperistalsis, lacrimation, salivation, and diaphoresis.
• Nicotinic Acetylcholine Receptors (nAChRs): Ligand-gated ion channels located at the neuromuscular junction (Nm) and autonomic ganglia/central nervous system (Nn). Moderate accumulation of acetylcholine enhances skeletal muscle twitch tension, whereas excessive, unceasing concentration induces depolarizing neuromuscular blockade, resulting in profound muscle fasciculation followed by flaccid paralysis.

The boundaries of anticholinesterase activity depend profoundly on whether the agent traverses the blood-brain barrier. Hydrophilic molecules possessing quaternary ammonium groups (such as neostigmine and pyridostigmine) carry a permanent positive charge, preventing penetration into the central nervous system and confining their pharmacodynamics to peripheral autonomic and somatic effectors. In contrast, lipophilic tertiary amines (such as physostigmine, donepezil, and rivastigmine) cross the blood-brain barrier readily, modulating cortical cognition, memory processing, and central autonomic regulation.

5. Historical Development

The history of anticholinesterase pharmacology bridges ethnographic discovery, foundational neurophysiology, and toxicological warfare. The earliest empirical encounters with anticholinesterase properties involved the Calabar bean (seeds of Physostigma venenosum), indigenous to West Africa. In local judicial customs, the beans served as “ordeal poisons” (esere). In 1846, Scottish medical missionaries witnessed these trials, prompting toxicologist Robert Christison and physician Thomas Richard Fraser to investigate the pharmacological actions of the bean in the 1850s and 1860s. Jobst and Hesse successfully isolated the active alkaloid physostigmine (also called eserine) in 1864. By 1877, Ludwig Laqueur demonstrated that topical physostigmine reduced intraocular pressure, inaugurating its therapeutic use in glaucoma.

The theoretical importance of cholinesterase inhibition became clear through the work of Otto Loewi and Sir Henry Dale. In 1921, Loewi identified Vagusstoff as the chemical mediator of vagal cardiac inhibition. Soon recognized as acetylcholine, its existence led Dale and colleagues to deduce that an extremely fast enzymatic inactivation must exist in tissues. In 1930, Edgar Stedman and his colleagues synthesized synthetic carbamates, discovering neostigmine, which exhibited greater chemical stability than physostigmine.

A transformative clinical milestone occurred in 1934 when Scottish physician Dr. Mary Broadfoot Walker recognized the physiological parallels between curare poisoning and the autoimmune pathology of myasthenia gravis. Walker administered physostigmine, and later neostigmine, to a myasthenic patient, demonstrating rapid reversal of ptosis and profound muscle weakness within minutes. This discovery represented one of the earliest successful applications of targeted molecular pharmacology in clinical neurology.

The dark chapter of anticholinesterase history emerged during the late 1930s. While investigating synthetic organophosphates as agricultural insecticides for IG Farben, German chemist Gerhard Schrader discovered the lethal potency of alkyl phosphates. This work culminated in the synthesis of the first modern organophosphorus nerve agents: tabun (1936), sarin (1938), and soman (1944). In the late 20th century, research pivoted toward the central nervous system, driven by the discovery of cholinergic deficits in Alzheimer’s disease. The United States Food and Drug Administration (FDA) approved tacrine in 1993, followed by second-generation centrally active inhibitors—donepezil (1996), rivastigmine (2000), and galantamine (2001)—which transformed neurocognitive pharmacology.

6. Theoretical Foundations

The conceptual application of anticholinesterases rests upon two major physiological models: the Katz Quantal Hypothesis of Neurotransmission and the Cholinergic Hypothesis of Age-Related Cognitive Dysfunction.

The quantal hypothesis, formulated by Sir Bernard Katz and colleagues in the 1950s, established that acetylcholine is packaged within presynaptic vesicles and released in discrete multimolecular units (quanta) upon calcium influx. At the skeletal neuromuscular junction, these packets bind post-junctional nicotinic receptors to generate miniature endplate potentials (mEPPs), which summate into a suprathreshold endplate potential (EPP) triggering muscle contraction. The anticholinesterase theoretical model posits that inhibiting post-junctional AChE widens the temporal and spatial diffusion radius of each released quantum. Consequently, non-depolarizing competitive receptor antagonists (such as rocuronium or tubocurarine) can be overcome by mass action, as elevated acetylcholine competes for unoccupied binding sites.

The cholinergic hypothesis, formalized by Bartus, Dean, Beer, and Lippa in 1982, asserted that the degenerative loss of cholinergic projections emanating from the basal forebrain (specifically the nucleus basalis of Meynert) to the neocortex and hippocampus constitutes a primary neurochemical correlate of cognitive and mnemonic decline in Alzheimer’s disease. Theoretical models suggested that while primary presynaptic cholinergic neurons undergo progressive apoptosis, residual functional synapses could be pharmacologically bolstered. Inhibiting acetylcholinesterase maximizes the biological utility of remaining acetylcholine release, compensating for diminished neuronal density without reversing the structural progression of underlying neuropathology.

7. Key Components, Types & Dimensions

Anticholinesterases are classified according to their chemical structures, selectivity profiles, and reaction kinetics:

• Non-Covalent Reversible Inhibitors:
  • Donepezil: A piperidine-based, highly selective, reversible inhibitor of AChE with low affinity for BChE. Primarily targets the central nervous system.
  • Edrophonium: A synthetic quaternary ammonium compound that binds reversibly via electrostatic interactions to the catalytic site. Possesses an extremely rapid onset and short duration of action (5–15 minutes).
  • Galantamine: A tertiary isoquinoline alkaloid exhibiting dual actions: competitive, reversible AChE inhibition and positive allosteric modulation of nicotinic acetylcholine receptors.
• Carbamate-Based (Pseudo-Irreversible) Inhibitors:
  • Neostigmine and Pyridostigmine: Quaternary ammonium carbamates that transfer a carbamyl moiety to the Ser203 hydroxyl group at the esteratic site. Carbamylated enzyme hydrolyzes far slower (hours) than the native acetylated enzyme (milliseconds). Impermeable to the blood-brain barrier.
  • Physostigmine: A lipophilic tertiary amine carbamate that penetrates the central nervous system, used as an antidote for severe central anticholinergic toxicity.
  • Rivastigmine: A centrally acting carbamate that inhibits both acetylcholinesterase and butyrylcholinesterase, dissociating slowly over approximately 8 to 10 hours.
• Irreversible Organophosphorus Compounds:
  • Pesticides: Compounds such as malathion, parathion, and chlorpyrifos. They phosphorylate the esteratic serine residue. Covalent dephosphorylation is so slow that return of biological function relies on de novo enzyme synthesis.
  • Chemical Warfare Agents: Volatile G-series (sarin, tabun, soman) and persistent V-series (VX) nerve agents. In addition to rapid covalent phosphorylation, these agents undergo a secondary unimolecular dealkylation reaction known as “aging.” Once aged, the enzyme-inhibitor complex cannot be reactivated by therapeutic oximes.

8. Examples & Illustrative Cases

To understand the clinical diversity of anticholinesterase agents, three classical illustrative scenarios across different clinical domains are presented below:

Case 1: Cognitive Stabilization in Mild-to-Moderate Alzheimer’s Disease
A 74-year-old retired architect presents with progressive episodic memory impairment, spatial disorientation, and difficulty completing multi-step instrumental activities of daily living. Neuropsychological evaluation demonstrates impairment on standardized memory recall tasks, and structural magnetic resonance imaging reveals bilateral hippocampal volume reduction without ischemic infarction. The patient receives a diagnosis of mild Alzheimer’s disease and begins treatment with donepezil at 5 mg daily, titrated after six weeks to 10 mg daily. Over the subsequent six months, the patient’s Montreal Cognitive Assessment (MoCA) score stabilizes, showing modest improvement in attention and executive processing due to elevated central cholinergic transmission.

Case 2: Autoimmune Myasthenia Gravis Management
A 32-year-old woman presents with fluctuating, fatigable bilateral ptosis and binocular diplopia that worsen toward the evening, alongside proximal upper-extremity weakness during sustained exertion. Serological testing confirms high titers of anti-acetylcholine receptor (AChR) antibodies. To restore functional neuromuscular communication, she begins therapy with the peripherally acting carbamate pyridostigmine bromide (60 mg administered orally every four hours while awake). Pyridostigmine inhibits peripheral AChE, allowing naturally released acetylcholine to remain active longer at the endplate, overcoming the autoimmune loss of viable nicotinic receptors and rapidly improving muscle strength.

Case 3: Acute Agricultural Organophosphate Toxicity
A 45-year-old farmworker is transported to an emergency department following accidental dermal exposure to concentrated parathion insecticide. The patient is found comatose, exhibiting profound miosis (pinpoint pupils), copious oral and bronchial secretions, audible wheezing from severe bronchospasm, diffuse muscular fasciculations, urinary incontinence, and severe bradycardia (34 beats per minute). Emergency physicians identify acute cholinergic crisis. Immediate management consists of aggressive intravenous atropine titration to competitively block life-threatening muscarinic hyperactivity, followed by pralidoxime chloride (2-PAM) to chemically reactivate the phosphorylated AChE before irreversible covalent “aging” occurs.

9. Measurement & Assessment

The assessment of anticholinesterase activity and its clinical consequences involves direct biochemical assays, neurophysiological monitoring, and structured functional evaluations.

The international reference standard for measuring cholinesterase inhibition in biological fluids is the Ellman Assay, developed by George L. Ellman in 1961. This spectrophotometric technique measures the cleavage of acetylthiocholine (or butyrylthiocholine) by cholinesterase into thiocholine and acetate. Thiocholine reacts instantaneously with 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB), yielding 5-thio-2-nitrobenzoate, a yellow dianion with peak absorbance at 412 nm. By quantifying absorbance changes per unit time, clinicians and toxicologists determine absolute enzymatic activity in red blood cell (RBC) cholinesterase (a direct surrogate for tissue AChE) and serum/plasma cholinesterase (butyrylcholinesterase or pseudocholinesterase).

In acute toxicology, RBC acetylcholinesterase inhibition reflects exposure severity: a reduction to 20–50% of personal or population baseline signals moderate toxicity, whereas depression below 10–20% correlates with life-threatening cholinergic crisis. In critical care settings, serum cholinesterase assays also monitor recovery following organophosphate or carbamate ingestion.

In neuromuscular and anesthetic applications, assessing anticholinesterase effects involves electrophysiological neuromuscular monitoring using Train-of-Four (TOF) stimulation of the ulnar nerve. Recovery from non-depolarizing neuromuscular block is confirmed when the fourth twitch amplitude matches the first (TOF ratio ≥ 0.90), demonstrating that anticholinesterase-induced acetylcholine accumulation has restored physiological somatic transmission.

10. Applications & Practical Significance

The applications of anticholinesterases span several clinical and biological disciplines:

• Neurology and Psychiatry: Reversible central inhibitors (donepezil, rivastigmine, and galantamine) serve as standard-of-care pharmacotherapy for mild, moderate, and severe Alzheimer’s disease, as well as Parkinson’s disease dementia and Lewy body dementia. They stabilize cognitive decline, improve global functioning, and reduce neuropsychiatric symptoms such as apathy and visual hallucinations.
• Anesthesiology and Perioperative Medicine: Following surgery performed under general anesthesia with non-depolarizing neuromuscular blocking drugs (such as rocuronium, vecuronium, or cisatracurium), intravenous neostigmine is administered in conjunction with an antimuscarinic agent (glycopyrrolate or atropine). This combination accelerates the clearance of paralysis while shielding the cardiovascular system from profound bradycardia and asystole.
• Neuromuscular Diseases: Pyridostigmine and neostigmine are essential long-term symptomatic therapies for autoimmune myasthenia gravis, Eaton-Lambert syndrome, and congenital myasthenic syndromes. Historically, the short-acting agent edrophonium formed the basis of the diagnostic “Tensilon Test,” evaluating rapid reversal of ptosis.
• Ophthalmology: Echothiophate iodide, an irreversible organophosphate solution, and physostigmine ointments are applied topically to manage refractory open-angle and angle-closure glaucoma. They contract the ciliary muscle, open trabecular meshwork pores, and enhance aqueous humor outflow.
• Clinical Toxicology: Physostigmine serves as a specific molecular antidote to reverse severe central and peripheral anticholinergic delirium induced by deliberate or accidental toxicity from drugs like diphenhydramine, tricyclic antidepressants, and scopolamine, or plants such as Datura stramonium.

11. Research & Empirical Evidence

Extensive clinical and translational investigations have evaluated the efficacy and biological actions of anticholinesterases. Large Cochrane Systematic Reviews examining donepezil, rivastigmine, and galantamine across dozens of randomized, double-blind, placebo-controlled trials have demonstrated statistically significant, albeit clinically modest, benefits. Patients receiving active therapy consistently maintain superior scores on the Alzheimer’s Disease Assessment Scale–Cognitive Subscale (ADAS-Cog) and Mini-Mental State Examination (MMSE) relative to placebo controls over treatment periods of 6 to 12 months. Evidence demonstrates that discontinuation commonly precipitates cognitive and functional decline that may not fully recover following drug reinitiation.

Beyond simple enzymatic inhibition, empirical findings highlight secondary neuroprotective properties. Anticholinesterases stimulate non-amyloidogenic amyloid precursor protein (APP) processing via activation of the alpha-secretase pathway, shifting processing away from the neurotoxic amyloid-beta cascade. Furthermore, basic research led by Kevin J. Tracey and colleagues has uncovered the “cholinergic anti-inflammatory pathway,” mediated largely through α7 nicotinic acetylcholine receptors located on peripheral macrophages and splenic lymphocytes. Anticholinesterases enhance this signaling axis, dampening systemic tumor necrosis factor-alpha (TNF-α), interleukin-1 (IL-1), and high mobility group box 1 (HMGB1) release in experimental models of endotoxemia and neuroinflammation.

12. Cultural & Cross-Cultural Considerations

Cultural context deeply influences human interaction with anticholinesterases. In West African history, the Calabar bean’s role as an ordeal poison rested upon religious and judicial frameworks: accused individuals who ingested the toxic brew were presumed innocent if they vomited the seeds (typically surviving due to rapid expulsion) and guilty if they succumbed to cholinergic crisis. Fraser’s isolation of physostigmine directly modernized colonial ethnobotany into Western laboratory pharmacology.

In modern global public health, organophosphorus anticholinesterases present an urgent socioeconomic challenge in low- and middle-income countries. Agricultural regions across South Asia, Southeast Asia, and Central America face widespread morbidity and mortality resulting from self-poisoning with organophosphorus insecticides. Research spearheaded by Michael Eddleston and colleagues in Sri Lanka has shown that pesticide self-harm accounts for hundreds of thousands of avoidable deaths annually. Global prevention frameworks prioritize strict legislative controls, community-level storage lockers, and pesticide bans (such as the phased elimination of monocrotophos and endosulfan), illustrating how chemical policy, socioeconomics, and medical pharmacology intersect.

13. Criticisms, Debates & Limitations

Despite their broad therapeutic adoption, anticholinesterases remain subject to significant clinical debate and therapeutic limitations. In dementia care, their benefits are primarily symptomatic rather than disease-modifying; they fail to arrest underlying tau aggregation, amyloid deposition, or widespread neurodegeneration. In many countries, health-technology assessment agencies (such as the UK’s National Institute for Health and Care Excellence, NICE) have debated the cost-effectiveness and real-world benefit of these agents relative to their adverse effect profiles.

Adverse effects represent a major clinical constraint. Elevated cholinergic tone frequently induces intolerable gastrointestinal disturbances, including nausea, vomiting, anorexia, weight loss, and severe diarrhea. Centrally active agents can disrupt sleep architecture, causing vivid dreams, insomnia, and nightmares. More critically, muscarinic stimulation of cardiac conducting tissue can induce sinus bradycardia, heart block, prolonged QTc intervals, and syncope, which increases fracture risk in elderly, fall-prone populations.

Another area of debate centers on the off-label use of anticholinesterases as “smart drugs” or nootropics among healthy individuals. While some non-sleep-deprived individuals experience subtle improvements in processing speed and working memory, others report anxiety, motor agitation, and gastrointestinal distress, showing that supra-optimal acetylcholine levels impair attention and working memory according to an inverted-U biological dose-response curve.

14. Related Terms & Distinctions

To avoid conceptual confusion, anticholinesterases must be clearly distinguished from related pharmacological classes:

• Acetylcholinesterase (AChE) vs. Butyrylcholinesterase (BChE): AChE is predominantly anchored to the post-synaptic membrane in neuronal and muscular tissues and localized on erythrocyte membranes, exhibiting high selectivity for acetylcholine. BChE (pseudocholinesterase), synthesized by hepatocytes and circulating freely in plasma, hydrolyzes butyrylcholine, succinylcholine, and diverse exogenous esters.
• Anticholinesterases vs. Direct Cholinergic Agonists: Anticholinesterases work indirectly by inhibiting endogenous neurotransmitter breakdown, requiring active baseline acetylcholine release. In contrast, direct cholinergic agonists (such as bethanechol, pilocarpine, and carbachol) bind and activate muscarinic receptors independently of endogenous neurotransmitter levels.
• Anticholinesterases vs. Anticholinergics: These are physiological antagonists. Anticholinesterases raise synaptic acetylcholine levels, whereas anticholinergics (such as atropine, scopolamine, and glycopyrrolate) competitively block muscarinic receptors, neutralizing anticholinesterase effects.
• Carbamates vs. Organophosphates: Carbamates undergo slower dissociation than native esters but eventually clear, earning them the label “pseudo-reversible.” Organophosphates form exceptionally stable, permanent covalent bonds that become refractory to oxime reactivators once chemical aging occurs.
• Pralidoxime (2-PAM): An oxime nucleophile that acts as an enzyme reactivator rather than an inhibitor. By attacking the phosphorus atom bound to the esteratic serine, it frees the enzyme from organophosphate inhibition.

15. Summary / Key Takeaways

Anticholinesterases represent an indispensable class of chemical agents operating at the core of cholinergic biology. By blocking acetylcholinesterase and butyrylcholinesterase, they sustain acetylcholine concentrations across somatic, autonomic, and central synapses. Their molecular designs span short-acting diagnostic compounds, therapeutic reversible carbamates and piperidines, and lethal organophosphates. In clinical medicine, they remain foundational therapies for managing Alzheimer’s dementia, stabilizing myasthenia gravis, reversing neuromuscular blockade in operating rooms, and treating life-threatening anticholinergic overdoses. In toxicology and public health, they require constant vigilance against agricultural poisonings and nerve agent threats. Balancing therapeutic benefit against cholinergic toxicity requires an exact understanding of their binding kinetics, receptor selectivity, and somatic versus central distribution.

References

• Bartus, R. T., Dean, R. L., Beer, B., & Lippa, A. S. (1982). The cholinergic hypothesis of geriatric memory dysfunction. Science, 217(4558), 408–414. https://doi.org/10.1126/science.7046051
• Birks, J. S. (2006). Cholinesterase inhibitors for Alzheimer’s disease. Cochrane Database of Systematic Reviews, 2006(1), CD005593. https://doi.org/10.1002/14651858.CD005593
• Colovic, M. B., Krstic, D. Z., Lazarevic-Pasti, T. D., Bondzic, A. M., & Vasic, V. M. (2013). Acetylcholinesterase inhibitors: Pharmacology and toxicology. Current Neuropharmacology, 11(3), 315–335. https://doi.org/10.2174/1570159X11311030006
• Eddleston, M., Buckley, N. A., Eyer, P., & Dawson, A. H. (2008). Management of acute organophosphorus pesticide poisoning. The Lancet, 371(9612), 597–607. https://doi.org/10.1016/S0140-6736(07)61202-1
• Ellman, G. L., Courtney, K. D., Andres, V., & Featherstone, R. M. (1961). A new and rapid colorimetric determination of acetylcholinesterase activity. Biochemical Pharmacology, 7(2), 88–95. https://doi.org/10.1016/0006-2952(61)90145-9