| 1. |
- Johannesson, Gunvor, et al.
(författare)
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Evaluation of an immunoaffinity extraction column for enrichment of adducts between human serum albumin and hexahydrophthalic anhydride in plasma.
- 2008
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Ingår i: Biomedical Chromatography. - : Wiley. - 0269-3879 .- 1099-0801. ; 22:3, s. 327-332
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Tidskriftsartikel (refereegranskat)abstract
- An immunoaffinity extraction (IAE) column was prepared for extraction of adducts between human serum albumin (HSA) and hexahydrophthalic anhydride (HHPA). HHPA is a strong sensitizer inducing immunoglobulin E antibodies in vivo. Polyclonal antibodies from a rabbit immunized with keyhole limpet hemocyananin-HHPA conjugate were purified using a Protein A Sepharose gel. To obtain antibodies with optimal affinity towards HHPA-protein adducts, HHPA-specific antibodies were selected using an N-hydroxysuccinimide-Sepharose column coupled with albumin-HHPA conjugate. Antibodies eluted from this column at pH 2.2 were selected to prepare the IAE column. The column was evaluated using 2 mL plasma spiked with HSA-HHPA conjugate. The column was eluted with glycine buffer at pH 2.0. The conjugates in the eluate were hydrolyzed to the corresponding HHP acid and quantified by mass spectrometry. The average recovery of HHPA adducts in 11 experiments was 68% with a coefficient of variation (CV) of 7%. The column's capacity to bind protein-HHPA adducts was found to be linear in the range of 0.15-1.2 nmol conjugate. The evaluation showed that the IAE column had adequate affinity towards the HHPA adducts and that the adducts could be extracted with good recovery and precision from a large volume of plasma.
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| 4. |
- Johannesson, Gunvor, et al.
(författare)
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Serum albumins are the major site for in vivo formation of hapten-carrier protein adducts in plasma from humans and guinea-pigs exposed to type-1 allergy inducing hexahydrophthalic anhydride
- 2001
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Ingår i: Clinical and Experimental Allergy. - 1365-2222. ; 31:7, s. 1021-1030
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Organic acid anhydrides (OAAs) are highly allergenic compounds used in the chemical industry. The OAAs probably act as haptens but the proteins that form conjugates with OAAs in vivo are still unknown. Conjugates between the anhydrides and serum albumins (SAs) have routinely been used when testing for OAA-specific antibodies. However, the use of SA as the carrier-protein in these tests has never been evaluated. OBJECTIVE: The aim of this study was to identify major and also immunologically relevant protein conjugates of a particularly sensitizing OAA, hexahydrophthalic anhydride (HHPA), in plasma. METHODS: Plasma was obtained from a HHPA-exposed worker, from a guinea-pig (GP) exposed to HHPA in an exposure chamber for 2 weeks (8 h/day, 5 days/week) and from a GP exposed once, nose-only, to tritium-labelled HHPA for 8 h. The plasma was fractionated using ion exchange chromatography and gel filtration. These fractions and also aliquots of unfractioned plasma were hydrolysed, derivatized and analysed for anhydride adduct content using gas chromatography-mass spectrometry. Further, plasma from the tritium labelled HHPA-exposed GP was separated by SDS gel electrophoresis and analysed by autoradiography. In addition, immunologically relevant proteins were identified through specific IgE and IgG immunoblottings using sera from exposed workers. RESULTS: For humans > 85% and for GPs > 74% of the HHPA-adducts coeluted with SA in plasma. Autoradiography of GP-plasma shows a single 66 kDa protein that binds HHPA. IgE immunoblotting shows a major 66 kDa and a minor 28 kDa protein which could be inhibited by HHPA-SA conjugate. IgG immunoblotting showed a major 66 kDa protein and several minor protein bands. CONCLUSION: This study shows SA to be the major protein in plasma that forms adducts in vivo with HHPA. The results also show that in an in vitro synthesized HHPA plasma protein conjugate, HHPA-specific IgE and IgG antibodies bind preferably to the SA.
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| 5. |
- Lindh, Christian, et al.
(författare)
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Analysis of chlormequat in human urine as a biomarker of exposure using liquid chromatography triple quadrupole mass spectrometry.
- 2011
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Ingår i: Journal of Chromatography. B. - : Elsevier BV. - 1873-376X .- 1570-0232. ; 879:19, s. 1551-1556
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Tidskriftsartikel (refereegranskat)abstract
- In this study, a method using liquid chromatography triple quadrupole mass spectrometry (LC/MS/MS) is described for the analysis of the plant growth regulator chlormequat (CCC) in human urine. Analysis was carried out using selected reaction monitoring (SRM) in the positive ion mode. [(2)H(4)] labeled CCC as internal standard (IS) was used for quantification of CCC. The limit of detection (LOD) was determined to 0.1ng/mL. The method was linear in the range 0.3-800ng/mL urine and had a within-run precision of 4-9%. The between-run precision was determined at urine levels of 7.0 and 31ng/mL and found to be 5 and 6% respectively. The reproducibility was 3-6%. To validate CCC as a biomarker of exposure, the method was applied in a human experimental oral exposure to CCC. Two healthy volunteers received 25μg/kg b.w. CCC in a single oral dose followed by urine sampling for 46h post-exposure. The CCC was estimated to follow a first order kinetic and a two compartment model with an elimination half-life of 2-3h and 10-14h respectively. One hundred 24h urine samples were collected from non-occupationally exposed individuals in the general population in southern Sweden. All samples had detectable levels above the LOD 0.1ng/mL urine. The median levels were 4ng/mL of CCC in unadjusted urine. The levels found in the population samples are several magnitudes lower than those found in the experimental exposure, which corresponds to an oral exposure of 50% of the ADI for CCC.
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| 6. |
- Lindh, Christian, et al.
(författare)
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Analysis of ethylenethiourea as a biomarker in human urine using liquid chromatography/triple quadrupole mass spectrometry.
- 2008
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Ingår i: Rapid Communications in Mass Spectrometry. - : Wiley. - 1097-0231 .- 0951-4198. ; 22:16, s. 2573-2579
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Tidskriftsartikel (refereegranskat)abstract
- Ethylenebisdithiocarbamates (EBDCs) are widely used fungicides. Ethylenethiourea (ETU), the main metabolite and also a contaminant in the commercially available products, is of major toxicological concern. In this study, a method using liquid chromatography/triple quadrupole mass spectrometry (LC/MS/MS) is described for the analysis of ETU in human urine after a single-step extractive derivatization using pentafluorobenzyl bromide (PFBBr). Analysis was carried out using selected reaction monitoring (SRM) in the positive ion mode. Quantification of ETU was performed using [(2)H(4)]-labeled ETU as internal standard (IS). The limit of detection (LOD) was determined to 0.05 ng/mL. The method was linear in the range 0.1-54 ng/mL urine and had a within-run precision of 3-5%. The between-run precision was determined at an average urine level of 2 and 10 ng/mL urine and found to be 9%. The inter-batch precision was 6%. To validate ETU as a biomarker of exposure, the method was applied in a human experimental oral exposure to the commercial fungicide Ridomil Gold, containing 64% mancozeb and 4.5% ETU. Two healthy volunteers received 8.9 microg/kg body weight (b.w.) Ridomil Gold in a single oral dose followed by urine sampling for 104 h post-exposure. The elimination half-life of ETU was estimated to 17-23 h.
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| 7. |
- Lindh, Christian, et al.
(författare)
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Binding of the potent allergen hexahydrophthalic anhydride in the mucosa of the upper respiratory and alimentary tract following single inhalation exposures in guinea pigs and rats
- 1999
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Ingår i: Toxicology. - 0300-483X. ; 134:2-3, s. 153-168
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Tidskriftsartikel (refereegranskat)abstract
- Hexahydrophthalic anhydride (HHPA; CAS No. 13149-00-3) is a highly allergenic compound commonly used in the chemical industry. Guinea pigs and rats were exposed to [3H2]HHPA by inhalation for 3-8 h and were killed at various intervals during 7 days. The tissue distribution of non-volatile and covalently bound radioactivity was studied by autoradiography. Tissue bound radioactivity was mainly found in the mucosa of the upper respiratory airways, whereas negligible levels were observed in the lungs. In addition, tissue bound radioactivity was present in the gastrointestinal tract and conjunctiva. Moreover, in the cortex of the kidneys in rats, but not in guinea pigs, a low level of tissue bound radioactivity was found. The radioactivity in the tissues persisted for at least 7 days after the end of exposure. Plasma proteins and soluble proteins from trachea, lung, and kidney from [3H2]HHPA-exposed animals were separated by gel filtration. The radioactivity in dialysed plasma was mainly found in the same fractions as albumin. The soluble proteins from trachea, lung, and kidney in both rats and guinea pigs showed a similar pattern as found in blood. The radioactivity in dialysed plasma from both guinea pigs and rats seemed to decay according to a two-compartment model. The non-extractable binding of [3H2]HHPA in the upper respiratory airways and conjunctiva may be of relevance for symptoms in workers with allergy, since they mainly develop symptoms and signs from the nose and eyes.
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| 8. |
- Rosqvist, Seema, et al.
(författare)
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Quantification of protein adducts of hexahydrophthalic anhydride and methylhexahydrophthalic anhydride in human plasma
- 2000
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Ingår i: Journal of Environmental Monitoring. - : Royal Society of Chemistry (RSC). - 1464-0325 .- 1464-0333. ; 2:2, s. 155-159
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Tidskriftsartikel (refereegranskat)abstract
- Hexahydrophthalic anhydride (HHPA) and methylhexahydrophthalic anhydride (MHHPA) are two highly allergenic compounds used in the chemical industry. A method was developed for quantification of protein adducts of HHPA and MHHPA in human plasma. The plasma was dialysed and the anhydrides were hydrolysed from the proteins at mild acidic conditions. The released hexahydrophthalic acid (HHP acid) and methylhexahydrophthalic acid (MHHP acid) were purified by reversed solid phase extraction followed by derivatisation with pentafluorobenzyl bromide. The derivatives were analysed using GC-MS in negative ion chemical ionisation mode with ammonia as moderating gas. As internal standards, deuterium labelled HHP and MHHP acids were used. The detection limits were 0.06 pmol mL(-1) plasma for HHP acid and 0.03 pmol mL(-1) plasma for MHHP acid. The between-day precisions for HHP acid were 18% at 0.3 pmol mL(-1) and 8% at 4 pmol mL(-1). For MHHP acid, the precisions were 13% at 0.3 pmol mL(-1) and 9% at 4 pmol mL(-1). There were strong correlations (r=0.94 for HHPA and 0.99 for MHHPA) between total plasma protein adduct concentrations and serum albumin adduct levels. Workers exposed to time-weighted average air levels of HHPA between < 1 and 340 microg m(-3) and between 2 and 160 microg m(-3) for MHHPA had plasma adduct levels between the detection limits of the methods and 8.40 and 19.0 pmol mL(-1), respectively.
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| 9. |
- Rosqvist, Seema, et al.
(författare)
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Total plasma protein adducts of allergenic hexahydrophthalic and methylhexahydrophthalic anhydrides as biomarkers of long-term exposure
- 2001
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Ingår i: Scandinavian Journal of Work, Environment and Health. - 0355-3140. ; 27:2, s. 133-139
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Tidskriftsartikel (refereegranskat)abstract
- OBJECTIVES: The aim of this study was to evaluate the applicability of total plasma protein adducts (TPPA) of 2 sensitizing low-molecular-weight allergens, hexahydrophthalic anhydride (HHPA) and methylhexahydrophthalic anhydride (MHHPA), as biomarkers of long-term exposure. METHODS: Urine samples from occupationally exposed workers were analyzed for the levels of urinary metabolites of HHPA and MHHPA, and the levels were used as the index of exposure. In addition, blood samples were obtained from the same persons, and the levels of TPPA were determined. Reversed solid phase extraction, derivatization using pentafluorobenzyl bromide, and gas chromatography-mass spectrometry analysis in the negative ion chemical ionization mode were used to quantify the exposure. To assess the suitability of TPPA as a biomarker of exposure to the anhydrides, the TPPA levels were correlated to urinary metabolite levels and hemoglobin (Hb) adducts. The toxicokinetics of TPPA were also studied to determine the elimination half-time of the adducts. RESULTS: The levels of TPPA correlated exceptionally well with the metabolite levels in the urine sampled repeatedly, giving r=0.97 for HHPA and r=0.92 for MHHPA. The TPPA of HHPA correlated highly with the Hb adducts with r=0.86. There were also good correlations between single urinary determinations and the TPPA levels (r(s)=0.71 and 0.81, respectively, for HHPA and MHHPA). The in vivo decay of TPPA gave an elimination half-time of 22 days for HHPA and 24 days for MHHPA. CONCLUSIONS: TPPA levels of HHPA and MHHPA are excellent biomarkers of long-term exposure to anhydrides.
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