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Ledowski T, Paech MJ, Clarke M, Schug SA, The influence of cate- cholamines on pseudocholinesterase enzymatic activity. Results of a laboratory investigation.
Journal of Clinical Monitoring and Computing (2006) 20: 329–332 DOI: 10.1007/s10877-006-9041-0

THE INFLUENCE OF CATECHOLAMINES ON PSEUDOCHOLINESTERASE ENZYMATIC ACTIVITY. RESULTS OF A LABORATORY INVESTIGATION Thomas Ledowski, MD, DEAA1, Michael J. Paech, FANZCA1,2, Michael Clarke, BSc3 and Stephan A. Schug, FANZCA1,2

Ó Springer 2006

Ledowski T, Paech MJ, Clarke M, Schug SA, The influence of catecholamines on pseudocholinesterase enzymatic activity. Results of a laboratory investigation. J Clin Monit Comput 2006; 20: 329–332

ABSTRACT. Objective. Acceleratory and inhibitory receptors have been described on the pseudocholinesterase (PCHE) molecule. An increased PCHE activity has been reported in patients with chronic pain and anxiety, conditions known to be correlated with increased plasma catecholamine levels. Aim of this laboratory investigation was to determine whether catecholamines have an effect on PCHE activity, as this knowledge could help to define the role of PCHE in the assessment of stress. Methods. After Ethics committee approval and written informed consent, 3 ml of blood was collected from five healthy volunteers. Fourteen samples of 50 ll each were prepared from each of the volunteerÕs plasma. Epinephrine (25, 50, 100, 200, 400 and 1000 pg) and norepinephrine (50, 100, 200, 400, 800 and 2000 pg) were added to samples of each subject. Sodium-chloride solution was added to control samples. PCHE activity was photometrically assessed. Results. PCHE activity was significantly higher after the addition of epinephrine (median 8304 versus 7386 U/l). This effect was not dose-dependent. PCHE activity did not change after addition of norepinephine. Conclusions. This mechanism might explain previous findings that showed higher levels of PCHE activity in the presence of chronic pain and anxiety. In the absence of a dose–response curve in the concentration range studied, PCHE activity does not appear to be suitable for the assessment of levels of stress. KEY WORDS. pseudocholinesterase, plasmacholinesterase, stress, catecholamines, epinephrine, norepinephrine.

INTRODUCTION

From the 1Department of Anaesthesia and Pain Medicine, Royal Perth Hospital, Wellington Street Campus, Perth, WA, 6000, Australia; 2School of Medicine and Pharmacology, The University of Western Australia, Perth, Western Australia, Australia; 3 Department of Core Clinical Pathology and Biochemistry, Royal Perth Hospital, Perth, Western Australia, Australia Received 12 May 2006. Accepted for publication 4 July 2006. Address correspondence to Thomas Ledowski, Department of Anaesthesia and Pain MedicineRoyal Perth Hospital, Wellington Street Campus, Perth, WA, 6000, Australia E-mail: [email protected]

An increase in pseudocholinesterase (PCHE) activity has been reported in association with preoperative anxiety [1]; anxiety related to other stressful events [2]; neuropsychiatric disorders [3]; and chronic pain [4]. It has also been suggested that measurement of the enzymatic activity of PCHE could be useful as a tool for the assessment of therapy of chronic pain [4] and anxiety [1]. The reason as to why enzymatic activity increases remains speculative. Previous authors have explained the rise in PCHE activity as a physiological hepatic response to an increase in the central release of acetylcholine, leading to acceleration of the production of PCHE [3, 4]. Alternatively, a direct interaction of intrinsic or extrinsic substances at the acceleratory binding site of the PCHE molecule has been postulated by Gero [5].

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Based on GeroÕs theory, and taking into account that anxiety and pain are likely to result in greater sympathetic outflow, a direct influence of stress hormones on PCHE could explain the observation of higher activity during anxiety and pain states. The primary aim of this laboratory investigation was to determine the influence of epinephrine and norepinephrine concentrations on PCHE activity. We postulated that, if a linear effect of increasing doses of catecholamines on PCHE activity was observed, the measurement of PCHE activity might serve as an indirect measure of stress. Thus the secondary aim of the study was to estimate a potential dose-effect response.

METHODS

After Ethics committee approval, and obtaining written informed consent from participants, 3 ml of blood was collected from five healthy volunteers. Blood samples were stored in lithium–heparin containers, centrifuged immediately and frozen at )80°C. Fourteen samples of 50 ll each were prepared from each of the volunteerÕs plasma, giving a total of 70 samples. One millilitre SARSTEDT tubes (SARSTEDT Australia Pty. Ltd.) were used with 500 ll 0.9% sodiumchloride solution added for predilution of each sample. Additionally, 5 ll 0.9% sodium-chloride solution was added to 10 samples (2 from each patient) to serve as controls. Epinephrine (epinephrine acid tartrate; Orion Laboratories, Welshpool, Australia), and norepinephrine (norepinephrine acid tartrate; Abbott Australasia, Kurnell, Australia) were diluted in 50 ml plastic containers containing 0.9% sodium-chloride solution to achieve the following concentrations: 5, 10, 20, 40, 80 and 200 pg/ll for epinephrine, and 10, 20, 40, 80, 160 and 400 pg/ll for norepinephrine. About 5 ll of each catecholamine concentration was added to 12 different samples from each subject to achieve in total, the addition of 25, 50, 100, 200, 400 and 1000 pg of epinephrine and 50, 100, 200, 400, 800 and 2000 pg of norepinephrine, respectively. The used range of catecholamine doses was chosen to imitate physiological plasma concentrations as reported during the perioperative period [6]. The samples were mixed and incubated at room temperature for 15 min, before PCHE activity was assessed automatically at 410 nm, 37°C and pH 7.7 (DATA PRO PLUS; Thermo Electron Corporation, Waltham, USA) using the photometric method (normal values of PCHE activity for this method 4300–12,400 U/l):

Butyrylthiocholine þ H2 O ! ðPseudocholinesteraseÞ ! Thiocholine þ butyrateThiocholine þ dithiobisnitrobenzoate ! 2Nitro5mercaptobenzoate Separate batches for both epinephrine and norepinephrine samples were run due to the limited maximum sample size, using five controls (one for each patient) and two internal controls per batch.

Statistical analysis The sample size estimation was based on the results of a previous study by Kambam et al. [7]. The alpha error was set at 0.05 and the beta error at 0.2. All variables were tested for normal distribution using the Kolmogorov–Smirnov test. As the data proved not to be normally distributed, the Wilcoxon test was used for comparison of the PCHE enzymatic activities. All data are described as median and 25%/75% percentiles.

RESULTS

PCHE activity was assessed in blood from a total of 5 subjects (2 female, 3 male). The internal, intra-assay quality control had a standard deviation of 149.89 U/l and a mean standard error of 83.53 U/l. Compared with control, PCHE activity was significantly higher after the addition of epinephrine (Table 1). The elevation of enzyme activity was first apparent after the addition of epinephrine 25 pg (median 8,304 versus 7,386 U/l), but did not increase further after larger doses

Table 1. Pseudocholinesterase (PCHE) activity in U/l (median [25%, 75% percentile]) after the addition of different doses of epinephrine Dose of epinephrine added (pg)

PCHE activity

(Control) 25 pg* 50 pg* 100 pg* 200 pg* 400 pg* 1000 pg*

7386 8304 8432 8050 8475 8304 8444

*Significantly different to control (p < 0.05).

[6127, [6853, [6950, [6717, [6957, [6804, [7006,

9309] 9914] 9935] 10043] 10113] 10200] 10340]

Ledowski et al.: Epinephrine Accelerates PCHE Activity

of epinephrine. The median difference between controls and samples with added epinephrine was 1089 U/L. After the addition of norepinephrine, PCHE activity did not change significantly (median change from control baseline: +106, +94, )299, )13, )299 and +48 U/l after addition of 50, 100, 200, 400, 800 and 1000 pg, respectively).

DISCUSSION

In this laboratory investigation, we demonstrated significantly higher PCHE activity after incubation of human plasma with epinephrine in concentrations that might be expected in human plasma. No changes were seen after the addition of norepinephrine. We were unable to demonstrate a dose-effect response in the range evaluated. The magnitude of PCHE activity increase was similar to what we found in a previous study that demonstrated significantly higher enzyme activity in preoperative patients compared with unstressed volunteers [1]. Our results support the findings of Swiergiel et al. [8], who reported increased activity of PCHE after the intramuscular injection of epinephrine into quails. In contrast, in 1948 Benson [9] found an inhibition of PCHE activity after the in-vitro addition of epinephrine. His, and our, study protocols were not comparable, however. He used lyophilized preparations of enzyme from a swine source and added epinephrine in concentrations several thousand times higher than those expected in human plasma [9]. The non-physiological amount of epinephrine might explain his results, because high doses of substances that activate PCHE can cause inhibitory effects on enzyme activity. This phenomenon was described by Ferko and Gero [10], who found a biphasic effect of morphine-derived compounds on PCHE activity. By performing an in-vitro study, we eliminated the possibility of any stress related or liver mediated influence on PCHE activity, as postulated by Cameron et al. [4]. Our results supported the theory of a direct molecular interaction between epinephrine and PCHE. An acceleratory PCHE receptor has been described for morphine-derived compounds [5]. Gero [5] concluded that the receptor contained two binding sites, at a distance of 6.0–6.5 A˚. He described the first as a hydrophobic cation-binding site, binding to a N-methyl group; and the second as a flat area accommodating a benzene ring. Our study design does not allow us to draw conclusions as to whether or not epinephrine binds at this particular site. Gero [5] postulated the need for a tertiary methylated amine group to enable substances to interact with the acceleratory receptor. Epinephrine has

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no such tertiary group, but contains a secondary methylated amine group, the presence of which might be responsible for an activation of PCHE. This postulate is supported by the absence of any effect from norepinephrine, which is almost identical to epinephrine, but lacks a methyl group. In our samples the increase of PCHE activity was first apparent after the addition of relatively small doses of epinephrine (25 pg) and did not increase further at higher epinephrine concentrations. This observation held not only for the median PCHE activity of all samples, but also within each individual subject. Therefore, it seems unlikely that different basic plasma levels of epinephrine in each of the five volunteers samples might have biased the results. In contrast, saturation kinetics for the interaction between epinephrine and PCHE could well explain our observations. Though we did not see any dose–response curve in the concentration range studied, we cannot exclude that there is one in concentrations