Annals of Occupational Hygiene Advance Access originally published online on January 7, 2005
Annals of Occupational Hygiene 2005 49(3):241-243; doi:10.1093/annhyg/meh086
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© 2005 British Occupational Hygiene Society Published by Oxford University Press;
Original Article |
Use of a Whole Blood Competitive Immunoassay for the Assessment of Worker Exposures to Propylene Oxide at Three Manufacturing Facilities
1 Lyondell Chemical Europe, Inc., Lyondell House, Bridge Avenue, Maidenhead, Berkshire SL6 1YP, UK; 2 Lyondell Chemie Nederland B.V., Europaweg 950, 3199 LC Maasvlakte Rt., P.O. Box 1050, 3180 AB Rozenburg ZH, The Netherlands; 3 Lyondell Chimie France SNC, Rte du Quai Mineralier, Zone Industrielle de Fos, 13270 Fos-sur-Mer, France
* Author to whom correspondence should be addressed. Tel: +44 1628 775 260; fax: +44 1628 775 263; e-mail: alan.jones{at}lyondell.com
ABSTRACT
The level of N-(2-hydroxypropyl)valine adducts in haemoglobin has been shown to correlate well with workplace exposure to propylene oxide (PO). However, the analytical method, using the modified Edman degradation procedure, is prohibitively time-consuming and expensive for use as a routine workplace exposure measurement tool. As an alternative, AB Biomonitoring Ltd of Cardiff, Wales, developed a competitive immunoassay for the determination of N-(2-hydroxypropyl)valine adducts in human haemoglobin. Studies showed that whole blood samples analysed using an enzyme linked immunosorbent assay (ELISA) and the modified Edman degradation procedure over the concentration range 3.7–992 nmol N-(2-hydroxypropyl)valine g–1 haemoglobin are in good agreement (correlation coefficient 0.998, n = 10). The intervariance and intravariance data indicate the repeatability of the ELISA method over the assay conditions employed and show that it is robust over its working range [2–200 pmol N-(2-hydroxypropyl)valine g–1 haemoglobin]. The assay employs a whole blood matrix and has a working range of 2–6000 pmol g–1 Hb (equivalent to up to 5 ppm PO exposure, 8 h per day, 5 days per week, over 4 months). The practicality of the assay was tested by assessing exposures to PO at three world-scale manufacturing sites in France and The Netherlands. Over 800 samples were taken over a 2 year period from operators, maintenance fitters and office staff. The data, typically <50 pmol g–1 globin, indicate that exposures were significantly <0.1 ppm at all times (The Dutch occupational exposure limit is 2.5 ppm over 8 h). Samples were taken after a major turnaround and also before and after the start-up of a newly commissioned plant. All data indicated that high levels of control were effective in minimizing exposure. This study has shown that the immunoassay is a powerful tool for the exposure component of future epidemiology studies, as well as a definitive demonstration of the effectiveness of exposure controls.
Keywords: biological monitoring • exposure measurement • haemoglobin adducts • immunoassay • propylene oxide
INTRODUCTION
Propylene oxide (PO) has a vapour pressure of 445 mmHg at 20°C and occupational exposure due to inhalation can occur. Dermal exposure may also occur during the production, storage, transport and use of PO. PO is classified by the International Agency for Research on Cancer (IARC) as a possible (2B) human carcinogen. Exposure should therefore be minimized, and the effectiveness of the measures taken can be assessed by means of biochemical effect monitoring.
The current occupational exposure limit (OEL) in the UK (maximum exposure limit, MEL) is 5 ppm, and in The Netherlands (MAC value) it is 2.5 ppm (6 mg m–3). Methods sensitive enough to measure <2.5 ppm are therefore required.
The modified Edman degradation procedure (Törnqvist et al., 1986
), used to determine N-alkylvalines, is a sensitive analytical method allowing the determination of N-(2-hydroxypropyl)valine adducts, even in non-occupationally exposed populations.
Adducts formed by the reaction of haemoglobin with small molecules such as PO are chemically stable and do not affect the average life span of erythrocytes of 
126 days to 4 months (Britton et al., 1991). Using the modified Edman degradation procedure, Boogaard et al. (1999) investigated adduct levels in potentially exposed workers and found a strong correlation between PO adduct concentration and time-integrated exposure, demonstrating the usefulness of adduct determination in occupational exposure monitoring and health surveillance programmes. Furthermore, Boogaard et al. calculated biological exposure limits (BELs) corresponding to an average level of exposure to an OEL over 4 months, 5 days per week, 8 h per day. For OELs of 5 ppm and 2.5 ppm, the BELs were 6400 and 3200 pmol N-(2-hydroxypropyl)valine g–1 globin, respectively.
Unfortunately, the determination of haemoglobin adducts using the modified Edman degradation procedure requires a complex work-up of samples prior to analysis and the use of sophisticated analytical equipment. These and other factors limit the usefulness of the method, particularly in large-scale screening programmes.
The use of immunoanalytical methods for the detection of biomarkers offers the prospect of an alternative approach (Van Welie et al., 1992). The introduction of simple, rapid and cost-effective immunoassays for determining haemoglobin adducts will allow the introduction of routine screening programmes.
Ball et al. (2004) confirmed the sensitivity and specificity of an assay using a polyclonal antiserum that recognizes N-(2-hydroxypropyl)valine. Laboratory comparisons indicated acceptable correlation with the Gas Chromatograph (GC)/Mass Spectrometry (MS) method. Therefore, a large-scale field study was carried out to test the practicality and acceptability of the method in the workplace, and to check conclusions from air monitoring that exposure controls were effective.
METHODS
Pre-sampling communications
Prior to the field study, each plant held a series of workplace meetings to explain the immunoassay technique and the implications of the presence of PO adducts in the blood of the workforce. All sampling was done on a voluntary basis, but very nearly 100% of the workforce agreed to participate in the study.
Sampling method
Ten millilitres of whole blood were taken from each operator by the site nurse and collected in normal Vacutainers with 1/3 EDTA (ethylene diamine tetraacetic acid) 15% as anticoagulant, either during a routine medical examination or during a specially arranged visit in cases where a medical had already been carried out. The vials were stored in refrigerators at 4–8°C.
Analytical method
The enzyme linked immunosorbent assays (ELISAs) were carried out by adding 50 µl of standard and 50 µl of antiserum diluted in phosphate-buffered saline/Tween (0.2%) to the central 60 wells of a 1 µg ml–1 bovine serum albumin N-(2-hydroxypropyl) heptapeptide conjugate coated plate. The plate was sealed with a plastic film and incubated overnight at room temperature. The contents of the wells were emptied, washed five times with saline/Tween (0.05%) wash solution and shaken dry. To each well was added 100 µl of an anti-rabbit immunoglobulin phosphatase conjugated antibody diluted 1:1000 in PBS/Tween (0.05%). The plate was covered and incubated for a further 2 h at room temperature. The plate was washed and dried, as above, and 100 µl of p-nitrophenyl phosphate (1 mg ml–1) in Tris buffer (0.2 M) was added to each well. In the presence of phosphatase this enzyme substrate turns from a colourless to a yellow solution. After 30 min the absorbance (405 nm) was measured using a Vmax microtitre plate reader (Molecular Devices).
Post-sampling communications
Only the medical staff had access to the sample codes linking the sample to an individual. Analytical data were therefore treated as medical information. Individuals were informed of their results by the medical unit, who provided the site industrial hygienist with the group data and any requested factor breakdowns but without individual profiles. All participants were informed that if they wished to discuss their own data with either the medical staff or the industrial hygienist, they were at liberty to do so.
All summaries of the data were discussed in workplace meetings by the industrial hygienist.
Data collection
Each plant used a different strategy.
- Plant A: Sampled all operators three times over 18 months. It also sampled maintenance fitters once during the year.
- Plant B: Sampled all operators once during the annual medical examination. It also sampled non-exposed personnel and carried out individual replication by taking two to four extra blood vials during the sampling. And it sampled all operators immediately after a full plant turnaround.
- Plant C: This was a new plant and all operators were sampled before start-up, and then 2 months after start-up.
- Plant B: Sampled all operators once during the annual medical examination. It also sampled non-exposed personnel and carried out individual replication by taking two to four extra blood vials during the sampling. And it sampled all operators immediately after a full plant turnaround.
RESULTS
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DISCUSSION
Using the Boogaard et al. (1999) correlation equation, 10 pmol g–1 globin of PO adduct is equivalent to an average airborne exposure of 0.008 ppm. The data in Table 1, therefore, indicate an extremely low level of PO exposure in all three Plants. Air sampling specifically on PO operators in Plants A and B had indeed shown very low levels of exposure not exceeding 0.1 ppm over 8 h (the limit of detection for this method was 0.01 ppm). It is not surprising, therefore, that the total operator workforce average is even lower than this level. The PO adduct data thus provide very powerful evidence that the controls in place in these plants are, in fact, successfully preventing any significant exposure to this material. The smoking/non-smoking comparison, shown in Table 2, confirmed that the PO content of cigarette smoke is not sufficient to be a confounding factor when examining occupational PO exposures.
From an acceptance and logistics standpoint, there was a very high voluntary participation both initially and in the subsequent repeats. That, plus verbal comments during the study, indicates that the method is not an onerous imposition and also that operators are enthusiastic about the use of a more personal approach to exposure assessment than the rather impersonal air sampling.
Future plans include using the PO biomarker as a routine medical blood analysis and also reducing the air monitoring and focusing only on tasks and places where changes are made.
Received June 10, 2004; in final form September 20, 2004
REFERENCES
Ball RL, Aston JP, Jones AL et al. (2004) Development of a competitive immunoassay for the determination of N-(2-Hydroxypropyl)valine adducts in human haemoglobin and its application in biological monitoring. BioMarkers 2004. (in print)
Boogaard PJ. (2002) Use of haemoglobin adducts in exposure monitoring and risk assessment. J Chromatogr; B 778: 309–22.
Boogaard PJ, Rocchi PSJ, Van Sittert NJ. (1999) Biomonitoring of exposure to ethylene oxide by determination of haemoglobin adducts: correlations between airborne exposure and adduct levels. Int Arch Occup Environ Health; 72: 142–50.[CrossRef][Web of Science][Medline]
Britton DW, Törnqvist M, Van Sittert NJ et al. (1991) Immunochemical and GC/MS analysis of protein and DNA adducts: dosimetry studies with ethylene oxide. In Gledhill BL, Mauro F, editors. New horizons in biological dosimetry. New York: Wiley Liss. pp. 79–88.
Törnqvist M, Mowrer S, Jensen S et al. (1986) Monitoring of environmental cancer initiators through hemoglobin adducts by a modified Edman degradation procedure. Anal Biochem; 154: 255–66.[CrossRef][Web of Science][Medline]
Van Welie RTH, Van Dijck RGJM, Vermeulen NPE et al. (1992) Mercapturic acids, protein adducts and DNA adducts as biomarkers of electrophilic chemicals. Crit Rev Toxicol; 22(5/6): 271–306.[Web of Science][Medline]
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