Activity of cholinesterases in a young and healthy middle-European population: Relevance for toxicology, pharmacology and clinical praxis
Introduction
Cholinesterases are polymorphic carboxylesterases displaying broad substrate specificity. There are two types named according to their localization in blood as red cell acetylcholinesterase (AChE; EC 3.1.1.7) and plasmatic butyrylcholinesterase (BChE; EC 3.1.1.8) (Taylor and Radić, 1994, Pohanka, 2011). AChE inhibition results in accumulation of the neurotransmitter acetylcholine in cholinergic synapses (autonomic nervous system, CNS and neuromuscular junctions) with typical signs of intoxication (Marrs, 1993, Maxwell et al., 2006, Peter et al., 2014). Acute BChE inhibition does not seem to cause critical features (Lockridge, 2015).
Both cholinesterases may be used as potentially stable biomarkers sensitive to selected inhibitors including organophosphorus nerve agents and pesticides (OPs) as well as some therapeutics (Munro et al., 1994, Kim et al., 2010, Ramírez-Santana et al., 2015, Cacciatore et al., 2015, Karasova et al., 2016). Among all the cholinesterase inhibitors, intoxication by organophosphorus pesticides is an important clinical issue around the World (Eddleston et al., 2002; Bertolote, 2006; McCauley, 2006; Gunnell et al., 2007). The symptoms of OP intoxication may be difficult to recognize in some cases when the etiology is unknown, so it is important to quantify a pre-exposure/baseline cholinesterase activity in the healthy population (Eddleston et al., 2008a, Peter et al., 2014). It is generally accepted that the degree of blood cholinesterase inhibition is related to the severity of the exposure and accompanying symptoms (bronchospasm, convulsions, vomiting, salivation, lacrimation, etc.). Thus, determination of the cholinesterase status in the case of intoxication is crucial for the early diagnosis of exposure to inhibitors and for further monitoring of the therapeutic efficacy of oxime (AChE reactivator) treatment (Eddleston et al., 2008b). These results are important for rapid and precise decision-making.
The measurement of peripheral cholinesterase activity may also be valuable in occupational medicine, and in diagnosis or prognosis in illness. The relationship between peripheral cholinesterase activity and brain injury/illness is most often explained by the activation of the “cholinergic anti-inflammatory pathway”. In the case of traumatic brain injury (TBI), correlation was found between serum cholinergic parameters and TBI severity, infective status, survival, and cognitive and neurofunctional outcomes, providing useful diagnostic and prognostic insights into TBI (Zhang et al., 2015). Also, certain relationships between peripheral AChE activity and brain Aβ plaques have been proved in Alzheimer disease patients. The link between brain Aβ and the peripheral cholinergic activity may also be explained by the “cholinergic anti-inflammatory pathway” (Alkalay et al., 2013).
Unfortunately, there is a general confusion in interpretation of AChE and BChE assays. Some OPs and other cholinesterase-inhibiting compounds used in industry inhibit BChE preferably to AChE. Thus, BChE may be used as a more sensitive marker of poisoning despite the fact that BChE inhibition does not relate to poisoning severity at all (Eddleston et al., 2008a, Lotti, 2001). It is also a useful marker for assessment of the elimination process in the warm-blooded organism. In contrast to BChE, the red cell AChE is currently considered as the major marker of poisoning severity and the need for atropine/oxime treatment (Katalinic et al., 2015).
The variability in cholinesterase measurement assays (electrometry, pH-stat, radiometry and colorimetry) is another problem in the clinical result interpretation. The colorimetric Ellman's method which is broadly preferred in occupational health screening represents a rapid, simple and cheap assay (Ellman et al., 1961). However interpretation of the results is rather unreliable due to variability in the method routine, especially in sample preparation, time of incubation and disturbances by the sample matrix. An appropriate understanding of Ellman's method limitations is essential for precise interpretation of the results.
The main aim of this study was the assessment of “normal/baseline” AChE and BChE activity in a young and healthy population, with subsequent evaluation of several intra-population factors including sex, age and smoking. Cholinesterase activity evaluation in whole blood and plasma was carried out robustly via a modified Ellman's method (Worek et al., 1999). This method was previously proved to have a high sensitivity and applicability in the therapeutic monitoring of organophosphate pesticide-poisoned patients. Moreover, the effect of two substantial factors – individual hemoglobin (Hb) levels and temperature during the in vitro assessment of cholinesterase activity was evaluated in this study. It was proved that both these factors have a significant impact on observed cholinesterase activity, and their importance for this methodology is considered.
Finally, the presented results might be useful in routine clinical practice where the monitoring of blood AChE and plasma BChE activity is crucial for prognosis and diagnosis in occupational medicine or in mass casualty scenarios.
Section snippets
Chemicals
Phosphate buffered saline tablets, acetylthiocholine iodide (ASCh), butyrylthiocholine iodide (BSCh), 5,5́-dithio-bis-2-nitrobenzoic acid (DTNB, so-called Ellman's reagent), Triton X-100, ethopropazine hydrochloride and huperzine A were purchased from Sigma Aldrich (Prague branch, Czech Republic).
Reagents
Phosphate buffer (PB; 0.1 M, pH 7.4) was obtained by dissolution of one tablet in 200 ml purified water and stored refrigerated at 4 °C for up 5 days for further use. Reagent for hemolysis of whole blood
Hemoglobin levels
Mean Hb level in the healthy middle-European population was found to be 150 ± 13 g/l. There was a significant inter-sexual difference in Hb levels (F1,375 = 5.4, p < 0.001). Hb level in males was almost 14% higher than that in females (159 ± 9 vs. 140 ± 9 g/l). As we found a significant interaction effect for sex × smoking (p = 0.04), data for male and female subjects were analyzed separately. The level of Hb in males was slightly higher for smokers (161 ± 8 g/l) than for non-smokers (159 ± 10 g/l). In contrast,
Discussion
This study described a rapid and simple method for in vitro detection of AChE and BChE activity/inhibition that may be used for early diagnosis and treatment of OP-poisoned patients (Sinko et al., 2007, Karasova et al., 2009a, Karasova et al., 2009b). The standard procedure for detection of changes in AChE activity – the Ellman method – was used. Nevertheless, this common method has many disadvantages that lead to misinterpretation of cholinesterase activity results. The major limitation of the
Conflict of interest
The authors declare no conflict of interest.
Acknowledgment
The authors would like to thank the nursing team (Transfusion Department, University Hospital, Hradec Kralove) for skilled technical assistance during sampling. This study was supported by the Long-Term Development of Research Organization 00179906 (University Hospital in Hradec Kralove, Czech Republic) and Long-Term Developing Plan 1011 (Ministry of Defense, Faculty of Military Health Sciences, Hradec Kralove, Czech Republic).
The authors are grateful to Ian McColl MD, PhD for language
References (37)
- et al.
Pesticide poisoning in the developing world – a minimum pesticides list
Lancet
(2002) - et al.
Management of acute organophosphorus pesticide poisoning
Lancet
(2008) - et al.
A new and rapid colorimetric determination of acetylcholinesterase activity
Biochem. Pharmacol.
(1961) - et al.
Curcumin protects against cigarette smoke-induced cognitive impairment and increased acetylcholinesterase activity in rats
Physiol. Behav.
(2012) Review of human butyrylcholinesterase structure, function, genetic variants, history of use in the clinic, and potential therapeutic uses
Pharmacol. Ther.
(2015)Clinical toxicology of anticholinesterase agents in humans
Organophosphate poisoning
Pharmacol. Ther.
(1993)- et al.
Limitation of the Ellman method: cholinesterase activity measurement in the presence of oximes
Anal. Biochem.
(2007) - et al.
Mechanism of stereoselective interaction between butyrylcholinesterase and ethopropazine enantiomers
Biochimie
(2011) - et al.
The effect of temperature on the activity of acetylcholinesterase preparation from rat-brain
Neurochem. Int.
(1987)
Improved determination of acetylcholinesterase activity in human whole blood
Clin. Chim. Acta
Chronic exposure to cigarette smoke during gestation results in altered cholinesterase enzyme activity and behavioral deficits in adult rat offspring: potential relevance to schizophrenia
J. Psychiatr. Res.
Plasma acetylcholinesterase activity correlates with intracerebral beta-amyloid load
Curr. Alzheimer Res.
Normal range of human red blood cell acetylcholinesterase activity
Drug Chem. Toxicol.
Deaths from pesticide poisoning: a global response
Br. J. Psychiatry
Butyryl-cholinesterase is related to muscle mass and strength. A new biomarker to identify elderly subjects at risk of sarcopenia
Biomark. Med.
Predicting outcome using butyrylcholinesterase activity in organophosphorus pesticide self-poisoning
Q. J. Med.
Improved Ellman procedure for erythrocyte cholinesterase
Clin. Chem.
Cited by (19)
Serum butyrylcholinesterase as a marker of COVID-19 mortality: Results of the monocentric prospective observational study
2023, Chemico-Biological InteractionsPoint-of-care measured serum cholinesterase activity predicts patient outcome following severe burns
2021, BurnsCitation Excerpt :Thus, the data collected at ERA would reflect a valid non-transfused baseline BChE level. Thirdly, inter-individual variability of BChE activity is well documented and can be affected by various factors, including gender and several comorbidities [32–34]. Analysis of our study population revealed comparable BChE activity in females compared to males.
Pesticide exposures for residents living close to agricultural lands: A review
2020, Environment InternationalCitation Excerpt :However, it can be difficult to interpret these measurements appropriately. For instance, certain cholinesterase-inhibiting compounds can inhibit red cell acetylcholinesterase more than butyrylcholinesterase in plasma (Karasova et al., 2017). Moreover, the interpretation of results needs to clearly determine an individual’s baseline levels, using repeated measurements (Rohlman et al., 2019), or his/her “paraoxonase status” referring to PON1 function as a modulation factor of cholinesterase inhibition (Dardiotis et al., 2019).
Tuning drug release from polyoxazoline-drug conjugates
2019, European Polymer JournalCitation Excerpt :The measured half-lives were slightly different, but the trends remain the same. The slight difference is attributed to the different levels of butrylcholinesterase activity in blood [17]. Surprisingly, in a third experiment, a different result was obtained with POZ-20 K-buprenorphine (BUP) conjugates with the 3-propionate linker.