Measuring of Cholinesterase Activity in Blood and Tissues of Bird’s by Electrometric Method: Review

Abstract

Many diseases and signs of organophosphate poisoning share common symptoms with other illnesses. Misusing pesticides can lead to severe damage to both the environment and people’s health. Insects, birds and other animals are affected by the pesticide due to its effect on cholinesterase (ChE). This enzyme breaks down acetylcholine into smaller parts that accumulate in neuromuscular junctions and nerve terminals. In cholinergic toxicity, signs appear such as nicotine-like effects and muscarinic side effects in the nervous system. In accordance with this study, an apparatus for measuring ChE-activity in birds’ blood and tissues is presented. Literature regarding proper ChE-activity in both wild and hybrid birds was found by consulting the Mosul city library. Additionally, the electrometric method proved accurate and effective. This makes it a good method for exposing potentially exposed birds in the natural world while also creating a biological control to reduce environmental pollution by carbamates or organophosphates.

1. Introduction

Pesticides are released into the environment through a complex chain of events and can be transported through air, water, land or even within organisms affecting birds’ environment. The extent of movement and spread can be local or far-reaching and varies according to the types of pesticides and their impact on the issue of food security and the provision of food for the ever-increasing world population while ignoring its effects on the birds that mainly protect crops from agricultural pests such as insects, caused their death and a significant decrease in their numbers with insect resistance to agricultural pesticides. Different types of pesticides belonging to other groups have been produced. This poses substantial risks to human health, birds and the environment [1].

Exposure of wild birds to pesticides leads to reduced egg production, which leads to a significantly decreased in the birds [2, 3]. Organophosphorus and pyrethroid compounds are used in agriculture and veterinary medicine [4, 5]. To control pest-borne diseases [6, 7, 8]. Excessive use of pyrethroids and organophosphates can cause human and animal contamination [9, 10, 11, 12, 13]. Organophosphate poisoning occurs due to exposure or abuse [14, 15] and intoxication occurs due to inhibition of ChE activity, leading to accumulation of acetylcholine in nerve endings and the appearance of muscarinic, nicotine and CNS symptoms-related intoxication, eventually leading to death [16, 17] but the main target of pyrethroids is the sodium channel Causes delayed blockage of the channel, resulting in a continuous flow of sodium, and its effect on voltage-sensitive chloride channels. Resulting in decreased chlorine flow through depleted airway channels [18, 19].

Measuring ChE activity in tissue and blood helps to detect organophosphate and pyrethroid toxicity in the early stages of intoxication. A 25–30% decrease in ChE activity in plasma or erythrocytes of birds is evidence of exposure such compounds [5, 9]. There appear to be several methods for measuring the activity of ChE, namely Ellman’s [20, 21], radiometric [22] and electrometric [23].

The last method is simple, cheap and does not require complicated equipment (just a pH meter and a water bath). Several changes have been made to the method, such as: e.g., electrometry (Michael method), by using different buffer solutions (setup and structure), and the interaction of sample volume with different time and temperatures [24, 25]. Michael’s original method was only used in humans, so some modifications were made due to various natural variations in ChE activity in the blood and tissues of different animals [24]. The main changes were to increase the incubation temperature from 20 to 37°C to increase the blood volume of the sample at the time of interaction and to minimize the incubation time depending on the animal type to which this method was applied to sheep [6, 24, 26]. Electroassay for Measuring ChE Activity in Dairy Cows [27]. The method as electrometric was not utilized previously on Budgie for measuring the activity of (ChE). Also, there is no information on the normal values of the ChE activity in birds.

2. Materials and methods

Use the electrometric method to measure the activity of (ChE) in different birds. It is important for detecting contamination with organophosphorus and carbamate pesticides that cause the death of birds by ingesting pesticides through contaminated water or food, such as fruits or insects, causing them to die. It also affects their young, causing them to die. Adequate information on normal levels of cholesterol activity in birds.

The present work uses blood and tissue samples from different birds, such as brain, liver, and muscle, ranging in sample size from 30 to 35 g. Blood samples were collected from the jugular vein (guillotine decapitation) and placed into tubes containing EDTA. Plasma was separated from blood by centrifugation at 3000 rpm for 15 minutes (Model 80-1, China). Place the brain, liver and thorax in a clean, dry plastic bag with crushed ice until homogenized at 3 mL per 100 mg of weight in barbiturate-phosphate saline (pH = 8.1) with a hand mixer. This is a sample preparation method for the purpose of screening for cholinesterase (ChE)-activity in samples that It was taken from natural birds.

3. Measure as stipulated in the method as electrometric

Blood samples and homogenized tissue were used to measure normal ChE activity. Measurements were done on the same day using electrometry Figure 1 [27, 28], using a pH meter type (pH 025 M, US) to measure the pH 1 of the samples, and then using a water bath type (HH-2 Digital Lab Thermostatic Water Bath, China) for use. After incubation at 37°C for 30 min, samples were taken directly from the incubator and pH 2 was measured. Measure the change in acid function using the following equation:

Figure 1.

Steps for the determination of blood cholinesterase activity by a modified electrometric method.

$$\left(\mathrm{pH}2-\mathrm{pH}1\right)-\mathrm{\Delta pH}\text{of blank}=\mathrm{\Delta pH}/30min.\left(\mathrm{ChE}\text{activity}\right)$$E1

ChE activity in budgie plasma and homogenate tissues was accurately measured electrometrically based on the mean and standard deviation [29], and the following coefficient of variation:

$$\text{Coefficient}=\frac{\text{Standard deviation}}{\text{Mean}}\times 100$$E2

The true and false percentages of ChE activity in the brain and plasma tissues of Budgie birds were calculated. Two sections of the plasma and brain tissue samples were separated. The first section of the samples was used to test the (ChE) activity (as previously described plus 40 μl of DW), and the second portion of the samples contained 40 μl of quinidine sulfate (0.1%) for each sample. Samples were incubated for 10 minutes at 37°C to inhibit pseudo-(ChE) [21, 30]. Through certain inhibitors, quinidine sulfate can prevent pseudo-(ChE) in brain and plasma tissues [20, 31]. Based on pseudo-(ChE) activity, the total residual (ChE) activity in brain and plasma tissues was identified as follows:

$$\begin{array}{l}\left(\mathrm{ChE}\right)\text{activity}=\text{total}\left(\mathrm{ChE}\right)-\text{activity\hspace{0.17em}of\hspace{0.17em}real}\left(\mathrm{ChE}\right)\\ \left(\text{With}\mathrm{no}\text{quinidine}\right)\left(\text{following\hspace{0.17em}quinidine\hspace{0.17em}addition}\right)\end{array}$$E3

Dichlorvos 50% (produced in China by Shenzhen Horizon Industry Co. Ltd.) and deltamethrin 2.5% were used to assess the inhibition of activity (ChE) in the brain tissues of Budgie birds in vitro (Indogulf company, India).

After obtaining the brains of seven Budgie birds for each experiment and incubating the samples for 10 minutes at 37°C to inhibit activity, inhibitor (ChE) incubation was carried out with concentrations of 0 (control), 0.5, and 1 μM/L for the treated group of Dichlorvos and concentrations of 0, 5 and 10 μM/L for the group of Deltamethrin [32]. Before inhibition, the percentage was calculated as follows:

$$\begin{array}{l}\text{Inhibition}\%\left(\mathrm{ChE}\right)\text{activity}=\\ \frac{\begin{array}{l}\text{Activity of}\left(\mathrm{ChE}\right)\text{for control sample}\left(\text{with}\mathrm{no}\text{dichlorovos or deltamethrin}\right)-\\ \text{Activity of}\left(\mathrm{ChE}\right)\text{with Dichlorovos or deltamethrin}\end{array}}{\begin{array}{l}\text{Activity of}\left(\mathrm{ChE}\right)\text{for control sample}(\text{with no}\\ \text{dichlorovos or deltamethrin})\end{array}}\times 100\end{array}$$E4

4. Results

Table 1 shows the normal reference range values, 95% confidence interval and related statistics for plasma, brain, liver and pectoralis muscle ChE activities of the bird species studied. Using the technique of in vitro ChE inhibition for 10 min by 0.1% quinidine sulfate, the estimated percentages of true ChE activity in the plasma of the Quail, Large pin-tailed sand grouse, Starling, Rock dove (Columba livia), Local dove (Streptopelia Senegalensis) and Budgie birds were 77%, 69%, 71% and 73%, 75%, 75%, respectively (Table 2).

Individuals per species
	Cholinesterase activity (ΔpH/30 min)
	Plasma	Brain	Liver	Muscle	Reference
Quail
Mean	1.23	0.39	0.19	0.06	[33]
Standard Error	0.098	0.013	0.021	0.004
Standard Deviation	0.310	0.040	0.066	0.013
95% confidence Interval	1.008, 1.452	0.360, 0.418	0.124, 0.146	0.050, 0.068
Range	1.71–0.72 = 0.99	0.45–0.33 = 0.12	0.34–0.09 = 0.25	0.08–0.04 = 0.04
Sand grouse
Mean	1.81	0.37	0.06	0.07	[33]
Standard error	0.109	0.025	0.009	0.005
Standard deviation	0.345	0.081	0.029	0.016
95% confidence interval	1.512, 2.068	0.304, 0.434	0.037, 0.068	0.054, 0.077
Range	2.58–1.44 = 1.14	0.51–0.25 = 0.26	0.12–0.01 = 0.11	0.09–0.04 = 0.05
Starling
Mean	1.10	0.24	0.08	0.08	[33]
Standard Error	0.074	0.029	0.006	0.008
Standard deviation	0.235	0.093	0.019	0.025
95% confidence Interval	0.936, 1.272	0.174, 0.308	0.066, 0.081	0.057, 0.093
Range	1.56–0.84 = 0.72	0.38–0.12 = 0.26	0.11–0.04 = 0.07	0.13–0.04 = 0.09
Rock dove (Columba livia)
Mean	1.28	0.59	0.12	0.08	[34]
Standard Error	0.08	0.026	0.010	0.008
Standard deviation	0.256	0.084	0.030	0.025
95% confidence Interval	1.100, 1.466	0.525, 0.585	0.094, 0.138	0.062, 0.099
Range	1.70–0.85 = 0.85	0.75–0.45 = 0.28	0.17–0.07 = 0.10	0.14–0.05 = 0.09
Local dove (Streptopelia Senegalensis)
Mean	0.82	0.39	—	—	[35]
Standard error	0.012	0.014	—	—
Standard deviation	0.038	0.044	—	—
95% confidence Interval	0.527, 0.587	0.302, 0.432	—	—
Range	1.68–0.90 = 0.78	0.65–.28 = 0.37	—	—
Budgie birds
Mean	0.31	0.23	0.12	0.11	[36]
Standard error	0.005	0.003	0.003	0.002
Standard deviation	0.015	0.009	0.007	0.005
95% confidence interval	0.30–0.32	0.22–0.23	0.11–0.12	0.10–0.11
Range	0.34–0.30 = 0.04	0.24–0.21 = 0.03	0.13–0.11 = 0.02	0.11–0.10 = 0.01

Table 1.

The cholinesterase activity values listed in this table were found out by an electrometric method in the bird species Quail, sand grouse, Starling, Rock dove (Columba livia), Local dove (Streptopelia Senegalensis) and Budgie birds. N = 10.

Cholinesterase values are the mean ± SE of 10 plasma samples for each of bird species.

Bird	Variable	(ΔpH/30 minutes)	Activity (%)	Reference
Quail	Total cholinesterase	1.230 ± 0.093	100	[33]
	True cholinesterase*	0.950 ± 0.069	77
	Pseudo cholinesterase	0.280 ± 0.054	23
Large pin-tailed sand grouse	Total cholinesterase	1.810 ± 0.109	100	[33]
	True cholinesterase*	1.240 ± 0.076	69
	Pseudo cholinesterase	0.570 ± 0.126	31
Starling	Total cholinesterase	1.100 ± 0.074	100	[33]
	True cholinesterase*	0.780 ± 0.025	71
	Pseudo cholinesterase	0.320 ± 0.042	29
Rock dove (Columba livia)	Total cholinesterase	1.280 ± 0.080	100	[33]
	True cholinesterase*	0.930 ± 0.063	73
	Pseudo cholinesterase	0.350 ± 0.028	27
Local dove (Streptopelia Senegalensis)	Total cholinesterase	0.03 ± 0.83	100	[35]
	True cholinesterase*	0.03 ± 0.62	75
	*Pseudo cholinesterase	0.018 ± 0.21	25
Budgie birds	Total cholinesterase	0.31 ± 0.005	100	[36]
	True cholinesterase*	0.002 ± 0.255	75
	Pseudo cholinesterase	0.06 ± 0.006	25

Table 2.

Estimation of true cholinesterase activity as determined by the described electrometric method in the plasma of Quail, Sand grouse, Starling, Rock dove (Columba livia), Local dove (Streptopelia Senegalensis) and Budgie birds.

^*

Quinidine sulfate was used to inhibit pseudo cholinesterase activity in the plasma.

Cholinesterase values are the mean ± SE of 10 plasma samples for each of bird species.

5. Conclusion

We conclude from the foregoing that measuring cholinesterase activity in the blood (plasma) and tissues (brain, liver, and muscles) of wild birds and domestic birds that live with humans is very important through which we discover the values of cholinesterase activity in wild birds, which reveal to us the extent of environmental pollution with these pollutants. (Pesticides) and their effect on wildlife, especially endemic and migratory birds, which cause the sudden death of large numbers of birds when these birds migrate through countries that use pesticides randomly and unstudied. Protecting our environment from pollution with organic phosphorus pesticides and carbamate pesticides that cause the death of birds directly or indirectly by poisoning and killing the organisms that birds depend on for their food, which causes the death of birds and the loss of this great environmental wealth. Because it maintains the ecological balance and is also a natural agricultural pesticide.

Acknowledgments

For their assistance and completion of this work, the author is grateful to the Environmental Science Department of the College of Environmental Science and Technologies at Mosul University in Iraq.