A Method for Diagnosing Organophosphate Pesticide Exposure in Humans Using Liquid Chromatography Coupled to Tandem Mass Spectrometry

Abstract

Organophosphate (OP) pesticides are commonly utilized worldwide for agricultural purposes and pose a health threat through air, ground and water contamination. Here, we present a convenient method for diagnosing exposure to OP pesticides in humans. This immunoprecipitation method relies on extraction of butyrylcholinesterase (BChE), a biomarker of OP poisoning that adducts OP compounds, from human serum using agarose beads conjugated to anti-BChE antibodies. Extracted BChE was then digested with pepsin and analyzed for unadducted and OP-adducted peptides by high-performance liquid chromatography coupled to tandem mass spectrometry. To characterize and validate this method, pooled human plasma was exposed to parathion and dichlorvos to form diethoxyphospho, aged ethoxyphospho and dimethoxyphospho adducts with BChE. Untreated plasma was also analyzed for unadducted peptides. Additionally, samples were analyzed using Ellman’s assay to measure BChE functional activity. The percent inhibition of BChE was 53.5 ± 5.76 and 95.2 ± 0.37%, respectively, for plasma treated with parathion for 1 and 24 h. The percent inhibition was 97.2 ± 0.98% for plasma treated with dichlorvos for 1 h. The percent inhibition was 97.9 ± 0.41% when the plasma treated with parathion (for 1 and 24 h) and dichlorvos (for 1 h) were mixed. Individual adducts were quantified in a single chromatographic run. Untreated plasma contained 26.4 ± 1.87 ng/mL of unadducted BChE and no adducted peptides. In contrast, the plasma sample treated with both pesticides contained no unadducted BChE, but did contain 9.46 ± 1.10, 10.9 ± 0.98 and 14.1 ± 1.10 ng/mL of diethoxyphospho, aged ethoxyphospho and dimethoxyphospho peptides, respectively. The ability to identify and measure BChE and BChE adducts to parathion and dichlorvos is expected to be useful for diagnosing human exposure to multiple OP pesticides.

The possible methods of diagnosis and effects of exposure to organophosphate pesticides

“…[4] It is a rapid and inexpensive method that is easy to conduct, as it provides useful information about the occurrence of exposure. For easy access, it has been made into a mobile kit for analyzing fresh samples [9]. The high reliance of this method on the normal enzyme activity in order to obtain the decrease in its level is the main issue, also what has been mentioned being the inter-and intra-individual variation in ChE values renders the severity of exposure [4].…”

“…The high reliance of this method on the normal enzyme activity in order to obtain the decrease in its level is the main issue, also what has been mentioned being the inter-and intra-individual variation in ChE values renders the severity of exposure [4]. This makes it difficult to interpret the condition of the patient without pre-exposure measurements, which generally are not available for most encountered patients [9]. Another method is through measuring the urinary metabolites, in contrast less invasive and logistically simpler [4].…”

“…Despite the fact that those metabolites have a relatively short half-life, resulting in difficulties determining the result several days after exposure. Additionally, the metabolites being measured can form naturally, which measurements tends to be an overestimation of the exposures presenting false positives [9].…”

“…The method itself remained simple, with centrifugation applied to extract the sample and what has been obtained is easily transferrable to standard laboratories. And utilization of Protein G-coated agarose beads provided solid support to immunoprecipitated BChE from plasma [9].…”

“…As some have involvement in the detoxification of OP such as paraoxonase, its activity being modified by the oxidative stress caused by OP [8]. Recently, developments have occurred where OP exposure can be diagnosed through the extraction of butyrylcholinesterase(BChE) from human serum and measure the adducts after digested with pepsin [9]. Those methods will be analyzed further in the following paragraphs.…”