Development of diagnostics in the search for an explanation of aerotoxic syndrome

Abstract

Aerotoxic syndrome is assumed to be caused by exposure to tricresyl phosphate, an additive in engine lubricants and hydraulic fluids that is activated to the toxic 2-(ortho-cresyl)-4H-1,3,2-benzodioxaphosphoran-2-one (CBDP). Currently, there is no laboratory evidence to support intoxication of airline crew members by CBDP. Our goal was to develop methods for testing in vivo exposure by identifying and characterizing biomarkers. Mass spectrometry was used to study the reaction of CBDP with human albumin, free tyrosine, and human butyrylcholinesterase. Human albumin made a covalent bond with CBDP, adding a mass of 170amu to Tyr411 to yield the o-cresyl phosphotyrosine derivative. Human butyrylcholinesterase made a covalent bond with CBDP on Ser198 to yield five adducts with added masses of 80, 108, 156, 170, and 186amu. The most abundant adduct had an added mass of 80amu from phosphate (HPO(3)), a surprising result given that no pesticide or nerve agent is known to yield phosphorylated serine with an added mass of 80amu. The next most abundant adduct had an added mass of 170amu to form o-cresyl phosphoserine. It is concluded that toxic gases or oil mists in cabin air may form adducts on plasma butyrylcholinesterase and albumin, detectable by mass spectrometry.

Figure 1

A portion of a MALDI MS spectrum taken from a complete peptic digest of CBDP-modified human serum albumin. An aliquot of the complete digest was diluted 10-fold with 50% acetonitrile plus 0.1% trifluoroacetic acid, and 1 μl was applied to a MALDI target plate. The values shown indicate the monoisotopic masses for VRYTKKVPQVSTPTL at 1717 amu, LVRYTKKVPQVSTPTL at 1830.1 amu, VRYTKKVPQVSTPTL with an added mass of 170 on Tyr411 to give 1887 amu, and LVRYTKKVPQVSTPTL with an added mass of 170 on Tyr411 to give 2000.1 amu. The structure is for the 2000.1 amu peptide which carries o-cresyl phosphate attached to Tyr411. The accession number for human albumin in the NCBI database is gi:122920512.

Figure 2

MALDI post source decay fragmentation spectrum of the CBDP-labeled human serum albumin peptide LVRYTKKVPQVSTPTL ([M+H]⁺¹ = 2000.2 amu). The spectrum was for an HPLC purified fraction of a peptic digest. The masses are centered over the peaks to which they apply. The y-axis is expanded 6.7-fold for peaks between 0 and 1800 m/z. The 306.0 amu mass, enclosed in a box, indicates the Tyr immonium ion derived from o-cresyl phosphate, whose structure is shown. The 1830.2 mass is the parent ion minus the OP.

Figure 3

MS spectrum from a mixture of tyrosine and CBDP, after 15.5 hours of reaction. Peaks marked by masses represent the reactants and the reaction products. The inserted structures are consistent with the indicated masses. Masses found in a mass spectrum of the buffer/matrix alone are marked by an asterisk (*). Three prominent, unassigned masses (at 288.0, 348.0 and 394.0 amu) are marked by a pound sign (#).

Figure 4

Time course for the reaction of CBDP with tyrosine at pH 7.8. The circles indicate CBDP, the squares a tyrosine-CBDP ring-opened adduct, and the triangles o-cresyl phosphotyrosine.

Figure 5

A portion of a MALDI MS spectrum taken on a complete tryptic digest of CBDP-inhibited human BChE. BChE was reacted with a 40-fold excess of CBDP for 30 seconds, and the reaction was stopped by addition of an equal volume of acetonitrile. The sample was digested with trypsin and the complete digest was diluted 10-fold in 50% acetonitrile plus 0.1% trifluoroacetic acid before applying 1 μl to the MALDI target plate. The values shown indicate the monoisotopic masses. The active site peptide SVTLFGES₁₉₈AGAASVSLHLLSPGSHSLFTR has a mass of 2928.4. Five adducts on serine 198 (serine 8 in the peptide) were found. They have masses of 3008.3, 3036.5, 3084.5, 3098.3, and 3114.3. Adduct structures are associated with the indicated masses. The accession number for human BChE in the NCBI database is gi:116353.

Figure 6

MALDI post-source decay fragmentation spectrum of the human BChE active site peptide labeled at Ser198 with phosphate (monoisotopic mass of 3008.7 amu). The spectrum was taken from an offline HPLC purified fraction of the tryptic digest. The masses are centered over the peaks to which they apply. The y-axis is expanded 6.7-fold for peaks between 0 and 2500 m/z. The symbol Δ indicates ions that contain dehydroalanine in place of serine 198 due to loss of phosphate plus a molecule of water. The y22 ion at 2275.1 amu retains the phosphate on serine 198.

Figure 7

A low-energy, collision induced fragmentation spectrum of the active site peptide from BChE, labeled with o-cresyl phosphate (monoisotopic mass of 3098.3 amu). The tryptic digest of CBDP-modified BChE was separated offline by HPLC and the fraction containing the 3098 amu mass was infused into the QTRAP 4000 mass spectrometer, using static infusion. The spectrum is the sum of 500 scans. Annotated masses include a singly-charged b-ion series (b2-b4), a singly-charged y-ion series (y3-y18) and a doubly-charged y-ion series (y18-y25). The symbol Δ indicates ions that have dehydroalanine in place of serine due to beta-elimination of the OP (mass of OP plus mass of a molecule of water). All other, unannotated, major peaks could be accounted for as b- or y-ions minus water or minus amine, as a-ions, or as internal fragments.

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