Elsevier

Toxicology Letters

Volume 277, 5 August 2017, Pages 24-31
Toxicology Letters

Activity of cholinesterases in a young and healthy middle-European population: Relevance for toxicology, pharmacology and clinical praxis

https://doi.org/10.1016/j.toxlet.2017.04.017Get rights and content

Highlights

  • The modified Ellman's method was used for AChE and BChE assessment in 387 healthy individuals.

  • The effect of three main factors (sex, age and smoking) was evaluated.

  • Results may be used in military and occupational medicine.

  • Reference values for AChE and BChE activity was proposed.

Abstract

The activity of human cholinesterases, erythrocyte acetylcholinesterase (AChE; EC 3.1.1.7) and plasma butyrylcholinesterase (BChE; EC 3.1.1.8) represents an important marker when monitoring exposure to pesticides/nerve agents, and may also be used in occupational medicine in diagnosis and prognosis of some diseases. In this study “normal/baseline” AChE and BChE activity has been investigated in a young and healthy population, with subsequent evaluation of several intra-population factors including sex, age (categories 18–25, 26–35 and 36–45 years old) and smoker status.

The modified Ellman's method was used for enzyme activity assessment in 387 young and healthy individuals (201 males and 186 females aged 18–45). A significant inter-sexual difference in AChE and BChE activity was found (AChE: 351 ± 67 for males and 377 ± 65 for females, (μmol/min)/(μmol of hemoglobin), p < 0.001; BChE: 140 ± 33 for males and 109 ± 29 for females, μkat/l, p < 0.001; mean ± SD). Despite the finding that mean AChE activity somewhat decreased whereas BChE activity grew within the age categories of the tested subjects, no significant effect of age on cholinesterase activity was found (p > 0.05). Smoking influenced cholinesterase activity – AChE activity in smokers was elevated (approx. 3% in males; 8% in females) relative to that in non-smokers (p < 0.05). Smoking was found not to have any effect on BChE activity.

Reference values based on confidence intervals for AChE and BChE activity were established. 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 of organophosphate poisoning, in occupational medicine and in relevant mass casualty scenarios.

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

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