Over the past 20 years, flight-crew members (both commercial and military) have reported symptoms such as dizziness, nausea, disorientation, blurred vision, short term memory issues and tingling legs which have been associated with smoke or fumes from the jet engines that have entered the cabin area. A prime candidate for the causative agent in these exposures is tri-cresyl phosphate (TCP) 2
, a common additive in engine lubricants and hydraulic fluids [1
]. TCP is a mixture of various positional cresyl isomers (o, m, p-TCP). Tri-o-cresyl phosphate (TOCP) is more toxic than the meta and para forms, which are considered to be non-toxic [1
]. Isomers containing mono-o-cresyl phosphate are considered to be the most toxic, followed by the di-ortho and tri-ortho compounds [5
TCP is infamously associated with “ginger jake paralysis”, a condition that afflicted 20,000-50,000 people in the United States in 1930. The paralysis was caused by the use of tri-cresyl phosphate (containing principally tri-o-cresyl phosphate) as an adulterant in Jamaica Ginger, a medicinal alcohol extract of ginger that was used for stomach problems and commonly abused as an illicit source of alcohol during prohibition [6
]. Another major outbreak of tri-cresyl phosphate poisoning, involving 10,000 victims, occurred in 1959 in Morocco. This one was caused by cooking oil adulterated with aircraft hydraulic oil [7
]. Other outbreaks have been reported [7
], with the latest occurring in China in 1995 [9
Paralysis in these cases involved the extremities, principally the legs, and appeared one to two weeks after consumption of the TCP [6
]. These outbreaks all involved oral consumption of high doses of tri-o-cresyl phosphate. A high oral dose for humans is considered to be around 6.6 mg of TOCP/kg body weight [10
]. As a consequence of these poisonings, manufacturers of TCP reduced the level of o-cresyl phosphate isomers in their products, from 25-40% in the 1930s-1940s to 0.1-1.0% in the 1990s [1
]. Reduction in tri-o-cresyl phosphate levels was the primary focus, however the content of mono-o-cresyl phosphate remains a point of concern.
Tri-cresyl phosphate currently is used as an anti-wear and extreme pressure additive in lubricants and hydraulic fluids [1
]. It has been used as a plasticizer in lacquers and varnishes, as a flame retardant in plastics and rubbers, as a lead scavenger in gasoline, and as a hydrophobic additive in waterproofing materials [2
]. Except for its use as an additive in engine lubricants and hydraulic fluids most commercial applications of TCP had been discontinued by 2002 [2
Assuming that ortho isomers of TCP are the causative agents in the airline incidents, symptoms appear after relatively low dose exposure. Exposure is presumed to occur from breathing contaminated cabin air or by absorption through the skin from deposits of contaminated particulates coming from the cabin air [1
]. Lack of accurate data makes it difficult to assess the actual level of exposure that may have been involved [3
], but the absence of paralysis argues that exposure levels were not high. The range of potential exposure covers four orders of magnitude. High dose exposure would correspond to breathing an atmosphere containing 1300 mg of tri-o-cresyl phosphate per cubic meter, for 30 minutes, by a 70 kg individual. This corresponds to oral consumption of 6.6 mg TOCP per kg, which is sufficient to cause serious paralysis. The recommended safe exposure limit is considered to be breathing 0.1 mg of tri-o-cresyl phosphate per cubic meter, for 30 minutes, by a 70 kg individual [10
]. Neither of these estimates takes into consideration the variability in P450 enzyme levels among individuals, or the higher toxicity of the mono-ortho isomer of TOCP.
The increasing number of reports from flight-crew members complaining of ill health following incidents of cabin air contamination by engine fumes [2
] prompted us to explore ways to test for exposure to tri-cresyl phosphate. Our plan was to use adducts formed on selected peptides from human serum albumin and human butyrylcholinesterase as biomarkers of exposure. Previous studies with other organophosphorus agents (OP) have identified tyrosine-411 as the most reactive residue on serum albumin [12
], and it is generally accepted that serine-198 is the only residue in butyrylcholinesterase that reacts with OP. Peptides associated with these residues have already been successfully used for determination of in vivo
exposure to other organophosphorus agents (OP) [13
]. We anticipated that adducts formed upon exposure to TOCP would appear on these same peptides. However, to properly apply these biomarkers to TOCP exposure, we first needed to more fully understand the reactions that might be involved. To that end, we have examined the reactions of human butyrylcholinesterase and human serum albumin with the principal, toxic metabolite of TOCP: 2-(o-cresyl)-4H-1,3,2-benzodioxaphosphoran-2-one (CBDP) [16
We chose to work with CBDP rather than with TOCP because it is accepted that TOCP is activated to CBDP in vivo
]. Activation involves oxidation of TOCP to di-o-cresyl-o-(alpha-hydroxy)tolyl phosphate, in the liver [16
] followed by cyclization, with the aid of albumin, to yield CBDP in the serum (). Cyclization will occur spontaneously, but at 1/10 the rate of that catalyzed by albumin [18
The toxic effects of TOCP are generally attributed to CBDP. CBDP is known to react with the active site serine in serine esterases, lipases and proteases, forming a covalent o-cresyl phosphate adduct, and causing inhibition of catalytic activity [16
]. Such a reaction with neurotoxic esterase has been proposed as the causative event in “organophosphorus ester induced delayed neuropathy” (OPIDN) [20
]. OPIDN is the formal description for ginger jake paralysis.
We performed four studies in this report. 1) We examined the mass spectrum of CBDP to determine its purity and we annotated the mass spectral fragmentation pattern of CBDP so that we would have a better understanding of the mass spectral properties of the free compound. CBDP was found to be pure. 2) We used mass spectrometry to test whether or not CBDP could react with human serum albumin. It was found that an o-cresyl phosphotyrosine adduct was formed on Tyr411. 3) We reacted CBDP with free tyrosine in solution to test the general reactivity of CDBP with tyrosine. Ortho-cresyl phosphotyrosine was formed in a two step process involving a transient intermediate (a ring-opened form of the cyclic saligenin portion of CBDP-tyrosine). 4) We reacted CBDP with human butyrylcholinesterase and analyzed the tryptic peptides using mass spectrometry to confirm that the reaction of CBDP was with the active site serine in this serine hydrolase and that the reaction gave an o-cresyl-phosphate serine adduct. We found that the active site serine (Ser198) formed several CBDP related adducts, the major adduct actually being phosphoserine with o-cresyl-phosphoserine being a minor species.