Original Contribution

Prognostic value of human erythrocyte acetyl cholinesterase in acute organophosphate poisoning

Nozha Brahmi MD^a,*, Amel Mokline MD^a, Nadia Kouraichi MD^a, Hayet Ghorbel PhD^b, Youssef Blel MD^a, Hafedh Thabet MD^a, Abderrazek Hedhili PhD^b, Mouldi Amamou MD^a

^aDepartment of Intensive Care Unit and Clinical Toxicology, 1008 Montfleury, Tunis-Tunisia

^bLaboratory of Toxicology, 1008 Montfleury, Tunis-Tunisia

Received 10 March 2006; revised 13 May 2006; accepted 13 May 2006

Abstract Acute organophosphate poisoning (OPP) such as dichlorvos may be monitored by the measurement of the erythrocyte acetyl cholinesterase (EAChE) and the serum cholinesterase (SChE) activities. The aim of this study was to look at correlation between the severity of the OPP judged by certain parameters such as coma, hemodynamic disturbances, respiratory failure, and the decrease of cholinesterases enzymes including EAChE and SChE at admission. Cholinesterase activity was determined upon admission and then on days 3 and 15 in the morning. Clinical effects, EAChE, and SChE activities data were investigated in 42 patients with OPP aged of 29.6 F 11.8 years with acute Cholinergic crisis in all cases. They were comatose in 29% of cases, presenting both hypotension or shock and hypoxemia in 17% of cases. Fifteen of them (36%) required mechanical ventilation. The mean EAChE activity at admission was 24.3 F 11.6 lmol/mL per hour at 37 8C; it was 1260 F 2204 IU/L for SChE. There were no correlations between the EAChE and the SChE activities. The EAChE was decreased only in comatose patients and those presenting hypotension, hypoxemia, and bradycardia with a cutoff of 23.5 lmol/mL per hour at 37 8C. Death was observed in 2 patients with a deep decrease of the EAChE at 5 lmol/mL per hour at 37 8C in 1 case and 9 lmol/mL per hour at 37 8C in another. The kinetics of improvement of the EAChE activity below the cutoff showed the absence of statistical improvement of the EAChE activity on day 3 (16.6 F 9 vs 19.5 F 5.7 lmol/mL per hour at 37 8C); this improvement was remarkable on day 15 (16.6 F 9 vs 27.5 F 6.5 lmol/mL per hour at 37 8C, P = .0004). In summary, the marked decrease of EAChE activity appears in this study as prognostic factor in acute OPP, and coma, respiratory failure, hemodynamic disturbances, and death are associated with a decrease of the EAChE of less than 23.5 lmol/mL per hour at 37 8C.

D 2006

Introduction

Organophosphate poisoning (OPP) is a major problem worldwide, especially in Developing countries, with millions

* Corresponding author. Tel.: +216 71341665; fax: +216 71314 180.

E-mail address: nozha_brahmi@yahoo.fr (N. Brahmi).

of cases and hundreds of thousands of deaths occurring each year [1,2]. Overall case fatality ranges from 10% to 20% [3,4], particularly in rural areas. For example, in a recent study from China that examined a nationally Representative sample of 518 suicides, 62% of deaths were due to pesticide ingestion and only 27% to physical methods [5]. In Sri Lanka, pesticide deaths as a percentage of total deaths by

0735-6757/$ – see front matter D 2006 doi:10.1016/j.ajem.2006.05.009

poisoning represent 77% in 2001 [6]. Currently, most deaths are due to class II organophosphate (OP) (in particular, dimethoate and fenthion) [6]. In a prospective study including 802 patients with OPP, dimethoate or fenthion poisoning were more severe than chlorpyrifos poisoning, with respective death odds ratio of 3.5 and 2.6 [2].

In this prospective study, we check whether if there exists a relationship between the erythrocyte acetyl cholinesterase (EAChE) activity and the severity of the OPP judged on certain parameters such as coma, neurologic complications, respiratory failure, and hypotension requiring vasopressors. We look also at correlation with serum cholinesterase (SChE) activity.

Materials and methods

Setting and patients

We prospectively included all patients admitted for acute OPP on a period of 6 months from July to December 2004 in a 16-bed intensive care and toxicological unit. Children and pregnant women were excluded.

During the period of the study were collected demo- graphical factors (age, sex, and medical history), clinical characteristics (pupils diameter, heart rate [HR], respiratory rate, systolic blood pressure (SBP), Glasgow Coma Scale (GCS), required mechanical ventilation , and the duration of hospital stay. Hypotension is defined by SBP of 90 mm Hg or lower, hypertension by SBP above 140 mm Hg, bradycardia by an HR below 60 beats per minute, and tachycardia by a HR of 100 beats per minute or higher. Coma is defined as GCS below 9.

Laboratory findings (Serum glucose, urea, sodium, potassium, Creatine phosphokinase, amylase, lipase, arterial blood gas), were measured daily. Hyperamylasemia and hyperlipasemia are defined by a serum concentration 3 times higher than the normal value.

The EAChE and SChE activities were performed at admission (H0) then on days 3 and 15 in the morning.

Once OP exposure occurs, pralidoxime is administrated via intravenous route, minimally for 24 hours, as a bolus of 5 mg/kg, immediately followed by a dose of 50 mg/kg daily. Intravenous infusion of Atropine is indicated as 1 mg/h to achieve atropinization (drying of excessive secretions).

Measurements of enzyme activities

Blood was sampled by venipunction in 2 tubes, red top tube for determination of SChE activity, and with heparin for EAChE. After sampling, the first tube was centrifuged for 15 minutes at 400 g, and the serum was separated. The activity of SChE with substrate S-butyrylthiocholine iodide, and

5.5 dithiobis-2-nitrobenzoate was determined using the auto- matic analyzer Cobas 400 (normal value, 5000-12000 IU/L). The activity of EAChE was determined according the Vincent D and Seronzac method after water hemolysis of

blood and colorimetric reaction using hydroxamic acetyl acid with ferric salt and lecture at 520 minutes (normal value, 27-47 lmol/mL per hour at 37 8C).

Statistical analysis

We did primary data analysis in SPSS 11.0 (SPSS, Chicago, Ill). Continuous variables were expressed as means (FSD), and subgroups evaluated by the v² test. P b .05 was considered statistically significant. Correlations were deter- mined using both Pearson and Spearman rank methods. Receiver operating characteristic curves were constructed to display the effects of various cutpoint levels of EAChE and the SChE activity on their sensitivity and specificity for clinical features and several outcomes. This study was approved by the appropriate ethics hospital committee.

Results

Forty-two patients without any history of neurologic or respiratory disease were admitted for acute OPP, in a suicidal attempt. Their mean age was 29.6 F 11.8 years, with a delay of consultation of 2.8 F 3.4 hours. The ingested products were dichlorvos (Fattek) in 40 cases and dimethoate in 2 cases. All patients developed muscarinic syndrome including miosis, vomiting, diarrhea, bronchor- rhea, bradycardia at 49 F 6 beats per minute (Table 1). Nicotinic effects were noted less frequently (21.5%) with tachycardia at 113 F 17 beats per minute in 8 cases, hypertension at 165 F 13 mm Hg in 4 patients, and fasciculation in 3 cases. Fourteen patients (33%) developed encephalopathy including coma and confusion (Table 1). Seven patients presented hypotension or shock with a mean SBP of 78 F 25 mm Hg (Table 1).

Biologic examinations showed hypoxemia (Pao₂/Fio₂ b300) in 7 cases (17%), metabolic acidosis in 22 patients (52%), hyperamylasemia in 11 patients (26%), and hyper- lipasemia in 5 patients. The mean EAChE activity at admission was 24.3 F 11.6 lmol/mL per hour at 37 8C; it decreased to less than 27 lmol/mL per hour at 37 8C only in 30 patients (71.4%), whereas the SChE (H0) was decreased in 39 patients with a mean value of

Table 1 The most frequent clinical features of OPP
at admission
Clinical features	Frequency	Percentage
Miosis	41	98
Vomiting	32	76
Diarrhea	23	55
Bronchorrhea	18	43
coma	12	29
Bradycardia	8	18
Tachycardia	8	18
Hypotension/shock	7	17
Confusion	5	12

EAChE (lmol/mL per hour at 37 8C)	24.3 F 11.6	27-47
SChE (IU/L)	1260 F 2204	5000-12000
Hyperamylasemia (IU/L)	544 F 115	28-60
Hyperlipasemia (IU/L)	880 F 378	40-190
Hypoxemia (Pao2/Fio2	183 F 28	–
b300 mm Hg) Metabolic acidosis	19.7 F 2	24-26

1260 F 2204 IU/L. (Table 2). In 3 patients, the SChE was normal at admission because they consult within 10 minutes after intoxication; their SChE was significantly decreased on day 3.

Table 2 Biologic disturbances at admission

Mean level at Normal value in admission (H0) our laboratory

(HCO^— b22 mEq/L)

3

Fifteen patients (36%) required MV for neurologic or respiratory failure with a mean duration of 54.9 F 56 hours. Pralidoxime was infused in all patients with a mean dose of 3498 F1894 mg; atropine was administrated in 40 patients; the total dose was 7 F 9 mg.

No correlation was found between the EAChE and the SChE at admission (Fig. 1). The kinetics of EAChE activity in all patients showed nonsignificant improvement between the first and the third and 15th days: 24.3 F 11.6 lmol/mL per hour at 37 8C (H0) vs 24.1 F 8.7 lmol/mL per hour at 37 8C (day 3) and 26.6 F 6 lmol/mL per hour at 37 8C (day 15) (Fig. 2A); on the other hand, a significant difference of EAChE (H0) activity between comatose and

noncomatose patients (18.3 F 11.9 vs 26.9 F 10.6 lmol/ mL per hour at 37 8C, P = .03) has been noted; this difference disappeared on days 3 and 15 (Fig. 2B).

In noncomatose patients, the EAChE decreased from the admission to the 15th day; this difference was not statistically significant (Fig. 2B).

The analysis of the severity of clinical features, according to the EAChE activity, showed a relationship between neurologic hemodynamic disturbances and the EAChE (H0) activity. The EAChE (H0) cutoff was 23.5 lmol/mL per hour at 37 8C for coma, with a sensitivity of 0.79, a specificity of 0.70, and an accuracy of 0.72; for MV (sensitivity, 0.79; specificity, 0.70; and accuracy, 0.76); and for hypoxemia (sensitivity 0.80; specificity 0.60 and accuracy 0.74) (Fig. 3); It was 22.5 lmol/mL per hour at 37 8C for bradycardia (sensitivity, 0.75; specificity, 0.70; and accuracy, 0.73) and for hypotension (sensitivity, 0.72; specificity, 0.70; and accuracy, 0.76). (Fig. 3) The EAChE was profoundly decreased in 2 patients who died; it was 5 lmol/mL per hour at 37 8C in 1 patient and 9 lmol/mL per hour at 37 8C in another. Although, in the statistical relationship between neurologic hemodynamic disturbances and the EAChE (H0) activity, no correlations were found neither with nicotinic effects nor with muscarinic syndrome,

Fig. 1 Relationship between EAChE and SChE. Fig. 2 Kinetics of EAChE activity.

Fig. 3 Receiver operating characteristic curves for neurologic state and hemodynamic disturbances.

including miosis, bronchorrhea, diarrhea, vomiting, and the EAChE (H0). However, muscarinic signs lasted longer in patients with decreased EAChE (H0) activity less than

23.5 lmol/mL per hour at 37 8C.

The comparison between patients with EAChE (H0) activity less than 23.5 lmol/mL per hour at 37 8C and those with EAChE activity above 23.5 lmol/mL per hour at 37 8C showed a significant difference in atropine infusion dose (9 F 12 vs 3 F 2.5 mg, P = .05), in pralidoxime dose (3.9 F

2.5 vs 1.9 F 1.7 g, P = .004), in EAChE (H0) (16.6 F 9 vs

33.3 F 6, P = 4.10^–10), and in EAChE on day 3 (19.5 F 5 vs 29.4 F 8.7; P = .02) (Table 3); this difference was not significant on day15 (Table 3).

When we analyze the kinetics of improvement of the EAChE activity below the cutoff, we noted a linear regression expressed by the following equation: Y = 0.7095x + 16.94, where Y is in lmol/mL per hour at 37 8C and x is in day. What was also interesting to demonstrate was the

Table 3 Variation of biologic examinations and management according to the erythrocyte AChE activity at admission
	EAChE b23.5 (H0) (n = 20) (47%)	EAChE N23.5 (H0) (n = 22) (53%)	P
EAChE (H0) (lmol/mL per hour at 37 8C)	16.2 F 9	33.6 F 6	4.10–10
EAChE (day 3) (lmol/mL per hour at 37 8C)	19.5 F 5	29.4 F 8.7	.02
EAChE (day 15) (lmol/mL per hour at 37 8C)	27.5 F 6.5	28.3 F 4	NS
Atropine (mg)	9 F 12	3 F 2.5	.05
Pralidoxime (g)	3.9 F 2.5	1.9 F 1.7	.004

Fig. 4 Kinetics of regeneration of EAChE in decreased EAChE (H0) patients.

absence of statistical improvement of the EAChE activity on day 3 (16.6 F 9 vs 19.5 F 5.7 lmol/mL per hour at 37 8C),

with a remarkable regeneration on day 15 (16.6 F 9 vs 27.5

F 6.5lmol/mL per hour at 37 8C, P = .0004) (Fig. 4).

Pancreatitis as early complication was noted in 5 patients. One patient developed toxic encephalitis confirmed by an electroencephalogram, and another presented a polyneurop- athy, which resolved favorably within 7 days. Death was noted in 2 patients seriously poisoned with dichlorvos; this mortality was attributed to OP toxicity in one case and to septic shock in the other.

Discussion

Organophosphate insecticides inhibit acetyl cholinester- ase (AChE) and cholinesterase enzymes resulting in over- stimulation at cholinergic synapses [2]. The differences in the Clinical courses depend on the properties of the different OP insecticide and their effect on decrease of EAChE activity [7,8]. In our study, we confirm the absence of correlation between the EAChE and the SChE activity (Fig. 1). As known, SChE is subject to high degree of variation induced by hereditary deficiency of this enzyme, Liver dysfunction, malnutrition, Iron deficiency anemia, drugs such as cocaine, morphine, codeine, and succinyl- choline, making this enzyme a less-than-perfect biomarker for OPP if baseline levels are unknown in an individual [9,10]. In fact, Nouira et al [11] reported in their study including 30 patients that the SChE levels at admission have no prognostic value in acute OPP. It follows that measure- ment of EAChE, which reflects inhibition at muscarinic and nicotinic synapses, would yield a more reliable and clinically significant result with better correlation in terms of clinical presentation [10,12]. Besides, the detailed investigation of EAChE activity and neuromuscular func- tion in pesticide-Poisoned patients showed that EAChE may serve as a surrogate biomarker for synaptic AChE [13-15]. In our study, coma, MV, hypoxemia, bradycardia, and hypotension were associated with a decrease of EAChE

activity less than 23.5 lmol/mL per hour at 37 8C. Besides, death was significantly associated with a profound decrease of the EAChE activity. On the other hand, no correlations were found between muscarinic and nicotinic signs and EAChE activity. These results are in agreement with those previously published. In fact and as reported by Bissbort et al [10] and Lotti [16], central system nerves depression and early respiratory failure were associated with high decrease of EAChE. The EAChE decrease was correlated to high risks of later neurologic complications [12,17-19], pancreatitis [20], and mortality [21].

Severity can be than evaluated by neurologic disturbances based on different scores and investigations. In fact, the monitoring of GCS score was used by Grmec et al [22] as a marker of prognosis, which allows an early and effective triage in OPP patients. In the study of Thiermann et al [12], the investigation of neuromuscular transmission was used as a parameter which reflects the AChE status at the synaptic site. In another study, the authors concluded that brain single photon emission computed tomography is a highly sensitive diagnostic method, together with clinical symptoms and SChE activity, for monitoring the Clinical prognosis of OPP [23]. In our study, we can conclude that coma, hypoxemia, and hemodynamic disturbances are associated with a decrease of EAChE (H0) activity less than 23.5 lmol/mL per hour at 37 8C. The decrease of the EAChE (H0) was remarkable in comatose patients (16.2 F 9 lmol/mL per hour at 37 8C) with a depression of more than 50% of baseline value. However, in 9% of cases (4 patients) we have not observed any sign of severity in spite of the decrease of the EAChE (H0) less than 23.5 lmol/mL per hour at 37 8C; this can be explained by genetic variation [24].

What can be discussed in our study is the trend in

EAChE on days 3 and 15 in patients with EAChE (H0) above 23.5 lmol/mL per hour at 37 8C. In fact, this decrease was not statistically different in days 3 and 15 and may be explained by an inhibition of a large fraction of the enzyme in the early days of intoxication. According to our results, pralidoxime should be indicated only in severe patients who decreased really their EAChE. However, and because of the absence of randomized controlled studies (pralidoxime vs placebo), we have to demonstrate, in a prospective double- blind controlled study, the effect of pralidoxime on the regeneration of the EAChE by testing 2 doses (50 and 100 mg/kg per day) vs placebo. In fact, and according to the clinical experience, there is a doubt about the clinical relevance of oximes for any form of OPP, in particular, in the Senanayake study group, which reported no difference in outcome in Sri Lanka when pralidoxime was unavailable in their hospital [4].

Conclusion

The marked decrease of EAChE activity appears in this study as prognostic factors.in acute OPP. Coma, requiring

MV, hypoxemia, hypotension, and bradycardia are associ- ated with a decrease of EAChE less than 23.5 lmol/mL per hour at 37 8C. In the opposite, muscarinic and nicotinic signs give evidence on OP exposure or intoxication but cannot be used as prognostic factors.

Acknowledgment

The authors thank the Toxicological Laboratory and the School of Pharmacy for collaboration and invaluable support in this investigation.

References

1. Eddleston M, Karalliedde L, Buckley N, et al. pesticide poisoning in the developing world–a minimum pesticides list. Lancet 2002; 360:1163 – 7.
2. Eddleston M, Eyer P, Worek F, et al. Differences between organophosphorus insecticides in human self-poisoning: a prospective study. Lancet 2005;366:1452 – 9.
3. Gunnell D, Eddleston M. Suicide by Intentional ingestion of pesticides: a continuing tragedy in developing countries. Int J Epidemiol 2003;32:902 – 9.
4. Eddleston M, Szinicz L, Buckley NA. Oximes in acute organophos- phorus pesticide poisoning: a systematic review of clinical trials. QJM 2002;95:275 – 83.
5. Phillips MR, Yang G, Zhang Y, Wang L, et al. Risk factors for suicide in China: a national case-control psychological autopsy study. Lancet 2002;360:1728 – 36.
6. Roberts D, Karunarathna A, Buckley NA, et al. Influence of pesticide regulation on acute poisoning deaths in Sri Lanka. Bull World Health Organ 2003;81:789 – 98.
7. Wadia RS, Bhirud RH, Gulavanti AV, et al. Neurological manifes- tations of three organophosphate poisons. Indian J Med Res 1977;66: 460 – 8.
8. Yang D, Lu X, Zhang W, et al. biochemical changes in primary culture of skeletal muscle cells following dimethoate exposure. Toxicology 2002;174:79 – 85.
9. Hedhili A, Thabet H, Slim L, et al. Variation de l’activite’ cholineste’rasique et globulaire au cours des intoxications aux

organophosphore’s et pathologies aigues. Biochim Clin 1991;15: 805 – 8.

1. Bissbort SH, Vermaak WJH, Elias J, et al. Novel test and its automation for the determination of erythrocyte acethylcholinesterase and its application to organophosphate exposure. Clin Chim Acta 2001;303:139 – 45.
2. Nouira S, Abroug F, Elatrous S, et al. Prognosis value of serum cholinesterase in organophosphate poisoning. Chest 1994;106:1811 – 4.
3. Thiermann H, Szinicz L, Eyer P, et al. Correlation between red blood cell acetylcholinesterase activity and neuromuscular transmission in organophosphate poisoning. Chem Biol Interact 2005;157-158:345 – 7.
4. Worek F, Szinicz L, Thiermann H. Estimation of oxime efficacy in nerve agent poisoning: a kinetic approach. Chem Biol Interact 2005;157-158:349 – 52.
5. Arsov Z, Zorko M, Schara M. Inhibition of erythrocytes acethylcho- linesterase by n-butanol at high concentrations. Arch Biochem Biophys 2005;437:78 – 84.
6. Kolf-Clauw M, Jez S, Ponsart C, et al. Acetyl- and pseudo- cholinesterase activities of plasma, erythrocytes, and whole blood in male beagle dogs using Ellman’s assay. Vet Hum Toxicology 2000;42:216 – 9.
7. Lotti M. Clinical toxicology of anticholinesterase agents in humans. In: Krieger R.I, editor. Handbook of pesticide toxicology, 2nd ed. San Diego7 Academic Press; 2001. p. 1043 – 85.
8. Aygun D, Doganay Z, Altintop L, et al. Serum acethylcholinesterase and prognosis of acute organophosphate poisoning. J Toxicol Clin Toxicol 2002;40:903 – 10.
9. Brahmi N, Gueye PN, Thabet H, et al. Extrapyramidal syndrome as a delayed and reversible complication of acute dichlorvos organophos- phate poisoning. Vet Hum Toxicol 2004;46:187 – 9.
10. Moretto A, Lotti A. Poisoning by organophosphorus insecticides and sensory neuropathy. J Neurol Neurosurg Psychiatry 1998;64:463 – 8.
11. Brahmi N, Blel Y, Kouraichi N, et al. Acute pancreatitis subsequent to voluntary methomyl and dichlorvos intoxication. Pancreas 2005;31:424 – 7.
12. Tsao TC, Juang YC, Lan RS, et al. Respiratory failure of acute organophosphate and Carbamate poisoning. Chest 1990;98:631 – 6.
13. Grmec S, Mally S, Klemen P. Glasgow Coma Scale and QTc interval in the prognosis of organophosphate poisoning. Acad Emerg Med 2004;11:925 – 30.
14. Yilmazlar A, O? zyurt G. Brain Involvement in organophosphate

poisoning. Environ Res 1997;74:104 – 9.

1. Singh S, Verma M, Leelamma CO, et al. Red cell acetyl cholinesterase and plasma cholinesterase activity and Genetic variants of plasma cholinesterase in northwest Indian adults. Int J Clin Pharmacol 1997; 35:357 – 60.