Bisphenol A concentration in human saliva related to dental polymer-based fillings.

OBJECTIVES: The aims of this study were to quantify salivary concentrations of bisphenol A (BPA) and to assess if presence of dental composite fillings is associated with higher BPA levels in saliva. MATERIALS AND METHODS: Twenty individuals with six or more tooth surfaces filled with polymer-based dental materials (composite group) and 20 individuals without any polymer-based materials (control group) were included in the study. Saliva was collected in polypropylene tubes and stored at -80 °C before analysis. Concentration of free (unconjugated) and total bisphenol A was determined by liquid chromatography/mass spectrometry (LC/MS). Values below limit of detection (0.1 ng/mL) were set to one-half of the limit of detection. Mann-Whitney U test (one sided; the Exact Tests Option in IBM-SPSS version 21) was used for the statistical analyses. RESULTS: The concentration of BPA in saliva was very low. In the composite group, 8 of 20 samples had detectable concentrations of BPA. In the control group, 3 of 20 samples had detectable concentrations of BPA. Statistical analysis indicated that the concentration of unconjugated BPA was slightly higher in the composite group (p = 0.044) than in the control group. CONCLUSIONS: Presence of dental composites may be associated with slightly higher concentration of unconjugated BPA in saliva. However, additional studies using sensitive analytical methods are needed before firm conclusions can be drawn. Influence from other factors, like food intake and time of the day for saliva sampling, must be considered. CLINICAL RELEVANCE: The relative contribution of existing polymer-based dental fillings to total BPA exposure seems to be low.

Prenatal exposure to environmental chemical contaminants and asthma and eczema in school-age children.

BACKGROUND: Emerging evidence suggests that prenatal or early-life exposures to environmental contaminants may contribute to an increased risk of asthma and allergies in children. We aimed to the explore associations of prenatal exposures to a large set of environmental chemical contaminants with asthma and eczema in school-age children. METHODS: We studied 1024 mother-child pairs from Greenland and Ukraine from the INUENDO birth cohort. Data were collected by means of an interview-based questionnaire when the children were 5-9 years of age. Questions from the ISAAC study were used to define asthma, eczema, and wheeze. We applied principal components analysis (PCA) to sixteen contaminants in maternal serum sampled during pregnancy, including perfluoroalkyl substances (PFASs), metabolites of diethylhexyl (DEHP) and diisononyl (DiNP) phthalates, PCB-153, and p,p'-DDE. Scores of five principal components (PCs) explaining 70% of the variance were included in multiple logistic regression models. RESULTS: In a meta-analysis that included both populations, the PC2 score, reflecting exposure to DiNP, was negatively associated with current eczema (OR 0.71, 95% CI 0.52-0.96). Other associations were not consistent between the two populations. In Ukrainian children, the PC3 score (DEHP) was positively associated with current wheeze (adjusted OR 1.56, 95% CI 1.03-2.37), whereas the PC5 score, dominated by perfluorooctanoic acid (PFOA), was inversely associated with current wheeze (OR 0.64, 0.41-0.99). In Greenlandic children, a negative association of PC4 (organochlorines) with ever eczema (OR 0.78, 0.61-0.99) was found. CONCLUSIONS: We found limited evidence to support a link between prenatal exposure to environmental chemical contaminants and childhood asthma and eczema.

Menstrual cycle characteristics in fertile women from Greenland, Poland and Ukraine exposed to perfluorinated chemicals: a cross-sectional study.

STUDY QUESTION: Does perfluorooctane sulfonate (PFOS) and perfluorooctanate (PFOA) exposure disrupt the menstrual cyclicity? SUMMARY ANSWER: The female reproductive system may be sensitive to PFOA exposure, with longer menstrual cycle length at higher exposure. WHAT IS KNOWN ALREADY: PFOS and PFOA are persistent man-made chemicals. Experimental animal studies suggest they are reproductive toxicants but epidemiological findings are inconsistent. STUDY DESIGN, SIZE, DURATION: A cross-sectional study including 1623 pregnant women from the INUENDO cohort enrolled during antenatal care visits between June 2002 and May 2004 in Greenland, Poland and Ukraine. PARTICIPANTS/MATERIALS, SETTING, METHODS: Information on menstrual cycle characteristics was obtained by questionnaires together with a blood sample from each pregnant woman. Serum concentrations of PFOS and PFOA were measured by liquid chromatography tandem mass spectrometry. Multiple imputations were performed to account for missing data. The association between PFOS/PFOA and menstrual cycle length (short cycle: ≤24 days, long cycle: ≥32 days) and irregularities (≥7 days in difference between cycles) was analyzed using logistic regression with tertiles of exposure. Estimates are given as adjusted odds ratios (ORs) with 95% confidence intervals (CIs). MAIN RESULTS AND THE ROLE OF CHANCE: Higher exposure levels of PFOA were associated with longer menstrual cycles in pooled estimates of all three countries. Compared with women in the lowest exposure tertile, the adjusted OR of long cycles was 1.8 (95% CI: 1.0; 3.3) among women in the highest tertile of PFOA exposure. No significant associations were observed between PFOS exposure and menstrual cycle characteristics. However, we observed a tendency toward more irregular cycles with higher exposure to PFOS [OR 1.7 (95% CI: 0.8; 3.5)]. The overall response rate was 45.3% with considerable variation between countries (91.3% in Greenland, 69.1% in Poland and 26.3% in Ukraine). LIMITATIONS, REASONS FOR CAUTION: Possible limitations in our study include varying participation rates across countries; a selected study group overrepresenting the most fertile part of the population; retrospective information on menstrual cycle characteristics; the determination of cut-points for all three outcome variables; and lacking information on some determinants of menstrual cycle characteristics, such as stress, physical activity, chronic diseases and gynecological disorders, thus confounding cannot be excluded. WIDER IMPLICATIONS OF THE FINDINGS: The generalizability of the study results is restricted to fertile women who manage to conceive and women who do not use oral contraceptives when getting pregnant or within 2 months before getting pregnant. To our knowledge only one previous epidemiological study has addressed the possible association between perfluorinated chemical exposure and menstrual disturbances. Though pointing toward different disturbances in cyclicity, both studies suggest that exposure to PFOA may affect the female reproductive function. This study contributes to the limited knowledge on effects of exposure to PFOA and PFOS on female reproductive function and suggests that the female reproductive system may be affected by environmental exposure to PFOA. STUDY FUNDING/COMPETING INTEREST(S): Supported by a scholarship from Aarhus University Research Foundation. The collection of questionnaire data and blood samples was part of the INUENDO project supported by The European Commission (Contract no. QLK4-CT-2001-00 202), www.inuendo.dk. The Ukrainian part of the study was possible by a grant from INTAS (project 012 2205). Determination of PFOA and PFOS in serum was part of the CLEAR study (www.inuendo.dk/clear) supported by the European Commission's 7th Framework Program (FP7-ENV-2008-1-226217). No conflict of interest declared.

Isotope dilution ESI-LC-MS/MS for quantification of free and total Nε-(1-Carboxymethyl)-L-Lysine and free Nε-(1-Carboxyethyl)-L-Lysine: comparison of total Nε-(1-Carboxymethyl)-L-Lysine levels measured with new method to ELISA assay in gruel samples.

ESI-LC-MS/MS method with isotope dilution and SPE based on cation-exchange was developed for determination of free and total Nε-(1-Carboxymethyl)-L-Lysine (CML) and free Nε-(1-Carboxyethyl)-L-Lysine (CEL). The use of nonafluoropentanoic acid in mobile phase was omitted, SPE recoveries of 82±3% and 91±10% (n=6) for CML and CEL respectively and, calibration curves (R(2)>0.9985) were attained. The method was applied to gruel samples and LoQ for the method was 5 ng/ml, RSD <10% and accuracy was 115%. Total CML levels in the gruel samples varied from 103-408 mg/kg protein. Free CML levels which were 1000 times lower than total CML were three times higher than free CEL levels. CML in a gruel sample was 127±7, 84±9 and 253±28 mg/kg using the current ESI-LC-MS/MS, ELISA and GC-MS respectively. The described method has advantages over ELISA with respect to reproducibility and specificity and over GC-MS with respect to reproducibility.

Exposure to perfluorinated compounds and human semen quality in Arctic and European populations.

BACKGROUND: Perfluorinated compounds (PFCs) have been suspected to adversely affect human reproductive health. The aim of this study was to investigate the associations between PFC exposure and male semen quality. METHODS: PFCs were measured in serum from 588 partners of pregnant women from Greenland, Poland and Ukraine who provided a semen sample, using liquid chromatography tandem mass spectrometry. Perfluorooctane sulfonate (PFOS), perfluorooctanoic acid (PFOA), perfluorohexane sulfonic acid (PFHxS) and perfluorononanoic acid (PFNA) could be detected in >97% of the samples. The associations between levels of these compounds and semen volume, sperm concentration, total sperm count, motility and morphology were assessed. RESULTS: Across countries, sperm concentration, total sperm count and semen volume were not consistently associated with PFOS, PFOA, PFHxS or PFNA levels. The proportion of morphologically normal cells was 35% lower [95% confidence interval (CI): 4-66%) for the third tertile of PFOS exposure as compared with the first. A similar reduction was found in relation to increasing PFHxS levels. At the third PFOA exposure tertile, the percentage of motile spermatozoa was 19% (95% CI: 1 to 39%) higher than in the first. CONCLUSIONS: The most robust finding in the present study was the negative associations between PFOS exposure and sperm morphology suggesting adverse effects of PFOS on semen quality, possibly due to interference with the endocrine activity or sperm membrane function. It cannot be excluded that this association and the positive association between PFOA and semen motility, which was not consistent across countries, might represent a chance finding due to the multiple statistical tests being performed.

Exposure to cadmium and persistent organochlorine pollutants and its association with bone mineral density and markers of bone metabolism on postmenopausal women.

Environmental contaminants such as cadmium and persistent organochlorine pollutants have been proposed as risk factors of osteoporosis, and women may be at an increased risk. To assess associations between exposure to cadmium and two different POPs (2,2',4,4',5,5'-hexachlorobiphenyl CB-153, 1,1-dichloro-2,2-bis(p-chlorophenyl)-ethylene p,p'-DDE), on one hand, and bone effects, on the other, in a population-based study among postmenopausal (60-70 years) Swedish women with biobanked blood samples. The study included 908 women and was designed to have a large contrast of bone mineral densities, measured with a single photon absorptiometry technique in the non-dominant forearm. Biochemical markers related to bone metabolism were analyzed in serum. Exposure assessment was based on cadmium concentrations in erythrocytes and serum concentrations of CB-153 and p,p'-DDE. Cadmium was negatively associated with bone mineral density and parathyroid hormone, positively with the marker of bone resorption. However, this association disappeared after adjustment for smoking. The major DDT metabolite (p,p'-DDE) was positively associated with bone mineral density, an association which remained after adjustment for confounders, but the effect was weak. There was no evidence that the estrogenic congener (CB-153) was associated with any of the bone markers. In conclusion, no convincing associations were observed between cadmium and POPs, on one hand, and bone metabolism markers and BMD, on the other.

Menstrual cycle characteristics in European and Inuit women exposed to persistent organochlorine pollutants.

BACKGROUND: Previous inconsistent results suggest that menstrual cycles may be disturbed by exposure to polychlorinated biphenyls (PCBs) and 1,1-dichloro-2,2-bis (p-chlorophenyl)-ethylene (DDE). METHODS: Information on menstrual cycle characteristics were obtained by questionnaires, and PCB and DDE were measured in serum samples from a total of 1494 women from Greenland, Swedish fishermen's wives, and inhabitants of Warsaw in Poland and Kharkiv in Ukraine. RESULTS: No consistent effects of PCB and DDE exposure on menstrual cycle characteristics were observed across populations. Within populations, we observed increased risks of short cycles (< or =24 days) among Swedish fishermen's wives exposed to high levels of PCB [odds ratio (OR) 2.5, confidence interval (CI) 1.2-5.1], and increased risk of long cycles (> or =32 days) among Polish women exposed to high levels of DDE (OR 3.1, CI 1.1-8.6). However, in Greenland it seemed that high levels of PCB or DDE were protective against long menstrual cycles (OR 0.7 CI 0.5-0.96 and OR 0.7 CI 0.5-0.99, respectively). CONCLUSIONS: It is unlikely that exposure to PCB and DDE is a main cause of menstrual disturbances. Genetic differences or dietary factors may be involved in the non-homogenous associations of organochlorine exposure and menstrual cycle between countries.

Biological monitoring of exposure to 1,5-naphthalene diisocyanate and 4,4'-methylenediphenyl diisocyanate.

OBJECTIVES: Biological monitoring of occupational sensitizers, such as 1,5-naphthalene diisocyanate (NDI) and 4,4'-methylenediphenyl diisocyanate (MDI) is of high importance. In this study, 1,5-naphthalenediamine (NDA) and 4,4'-methylenedianiline (MDA) in hydrolysed urine and plasma were evaluated as biomarkers of exposure to NDI and MDI, respectively. METHODS: The air exposure to NDI and MDI was monitored for 30 exposed workers at four different plants. In parallel, urinary as well as blood plasma samples were collected. Biomarker levels were determined in hydrolysed urine and plasma by means of gas chromatography-mass spectrometry. RESULTS: Air exposure to both MDI and NDI was correlated to their corresponding urinary and plasma biomarkers. The correlation coefficients for the associations between air and biomarker levels were in the range of 0.51-0.65 and 0.53-0.96 for MDI and NDI, respectively. For NDI, but not for MDI, the significance and correlation coefficients were increased by adjusting the urinary biomarker levels for creatinine content or density. CONCLUSIONS: Biomarker and air levels of MDI and NDI were correlated, but there was a large individual variation.

Adducts between nucleophilic amino acids and hexahydrophthalic anhydride, a structure inducing both types I and IV allergy.

Haptens causing type I allergy have been shown to predominantly form lysine adducts in the carrier protein, while many haptens giving rise to type IV allergy preferentially form adducts with cysteine residues. Hexahydrophthalic anhydride derivatives are strong sensitizers capable of inducing allergic rhinitis, asthma and urticaria (type I allergy) and allergic contact dermatitis (type IV allergy). The ability of hexahydrophthalic anhydride (HHPA) to form adducts with nucleophilic amino acids and a model peptide in vitro is presented. Adduct formation was monitored by high-performance liquid chromatography with ultraviolet light/vis detection (LC-UV/vis) and high-performance liquid chromatography with mass spectrometric detection (LC/MS). The characterization was obtained by nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry (MS and MS/MS). It was found that HHPA formed adducts with N(alpha)-acetylated lysine and cysteine and the non-acetylated alpha-amino group of proline and, to some extent, also with other nucleophilic amino acids. The adducts with lysine and proline were chemically stable. Addition of one HHPA to a model carrier peptide with all important nucleophilic amino acid residues showed N-terminal proline to be the major site of reaction. The addition of a second hapten gave a lysine adduct, but a minor cysteine adduct was also found. The cysteine-HHPA adducts were shown to be chemically unstable and participated in further reactions with lysine forming lysine-HHPA adducts. The results will be useful for understanding the formation of HHPA-protein adducts with the capability of being markers of exposure, and also to a deeper understanding of the chemical structures causing types I and IV allergy.

Exposure to persistent organochlorine pollutants and seminal levels of markers of epididymal and accessory sex gland functions in Swedish men.

BACKGROUND: A major exposure route for persistent organochlorine pollutants (POPs) in Sweden is through consumption of fatty fish from the Baltic Sea. Endocrine disruptors, such as POPs, may have a negative impact on sperm quality. The present study aimed to investigate whether exposure to 2,2',4,4',5,5'-hexachlorobiphenyl (CB-153) and 1,1-dichloro-2,2-bis (p-chlorophenyl)-ethylene (p,p'-DDE) affects epididymal and accessory sex gland function. METHODS: 157 fishermen from the coastal stretches of Sweden, aged 27-67 years, provided semen samples which were analyzed for prostate-specific antigen (PSA), neutral alpha-glucosidase (NAG), fructose and zinc levels. Serum levels of CB-153 and p'p-DDE were determined. RESULTS: The median CB-153 serum level was 189 ng/g lipid (range 40-1460) and a median p,p'-DDE serum level 231 ng/g lipid (range 40-2252). There was a significant linear association between CB-153 and total amount of PSA (slope [beta] = -2.5, 95% confidence interval [CI] -4.0, -0.9; P = 0.02). With age, abstinence time and smoking included in the model the association became non-significant (beta = -1.4, 95% CI-3.0, 0.1; P = 0.07). There were no significant associations between CB-153 and zinc, fructose and NAG. As for the exposure variable p,p'-DDE and the outcome variables, no significant associations were found. CONCLUSIONS: The study gives only very limited support of an association between CB-153 in serum and total PSA, and a random finding cannot be excluded.

Identification and characterisation of adducts between serum albumin and 4,4'-methylenediphenyl diisocyanate (MDI) in human plasma.

Diisocyanates are potent inducers of airways disease. Methylenediphenyl diisocyanate (MDI) is a widely used diisocyanate in the chemical industry. The aim of this study was to identify major and also immunologically relevant protein conjugates of MDI in plasma. Plasma was obtained from an MDI-exposed worker. The plasma was dialysed and then fractionated using ion exchange chromatography (IEC) and gel filtration. These fractions and also aliquots of unfractioned plasma were hydrolysed, derivatised and analysed for isocyanate adduct content using gas chromatography-mass spectrometry. In addition, immunologically relevant proteins were identified through specific IgG immunoblotting using pooled sera from two exposed workers. It was shown by dialysis that 96% of the hydrolysed MDI derivatives were protein bound and that 95% of the MDI adducts co-eluted with serum albumin in plasma using IEC. All MDI-protein adducts co-eluted with serum albumin using gel filtration. IgG immunoblotting showed a major 66 kDa protein and also some intermolecular reactions in serum albumin. This study shows serum albumin to be the major protein in plasma that forms adducts in vivo with MDI. Thus, a quick and simple quantitative method for biological monitoring may be developed for MDI exposure. The results also showed that MDI-specific IgG antibodies preferentially bind to the serum albumin in in-vitro-synthesised MDI-plasma protein conjugates.

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.

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.

Total plasma protein adducts of allergenic hexahydrophthalic and methylhexahydrophthalic anhydrides as biomarkers of long-term exposure.

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.

Quantification of protein adducts of hexahydrophthalic anhydride and methylhexahydrophthalic anhydride in human plasma.

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.

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.

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.

Quantification method of human hemoglobin adducts from hexahydrophthalic anhydride and methylhexahydrophthalic anhydride.

A method was developed for the determination of human hemoglobin (Hb) adducts from hexahydrophthalic anhydride (HHPA) and methylhexahydrophthalic anhydride (MHHPA). The procedure includes lysis of erythrocytes, dialysis of the Hb-solution followed by acid hydrolysis. The released hexahydrophthalic (HHP) acid and methylhexahydrophthalic (MHHP) acid were purified using a combined liquid-liquid and solid-phase extraction procedure followed by derivatization with pentafluorobenzyl bromide. The derivatives were analyzed using GC-MS in negative ion chemical ionization mode with ammonia as moderating gas. As internal standards, deuterium-labeled HHP and MHHP acids were used. The detection limits were 0.3 pmol/g Hb for HHP acid and 0.9 pmol/g Hb for MHHP acid. The between-day precisions for HHP acid were 18% at 2 pmol/g Hb and 10% at 13 pmol/g Hb. For MHHP acid, the precision was 27% at 2 pmol/g Hb and 14% at 22 pmol/g Hb. The method was applicable for analysis of Hb adducts from workers occupationally exposed to HHPA and MHHPA.

Human hemoglobin adducts following exposure to hexahydrophthalic anhydride and methylhexahydrophthalic anhydride.

Hexahydrophthalic anhydride (HHPA) and methylhexahydrophthalic anhydride (MHHPA) are highly allergenic compounds used in the chemical industry. The aim of this study was to characterize the protein adducts in erythrocytes following exposure to HHPA and MHHPA. Blood and urine samples were obtained from 51 HHPA- and MHHPA-exposed workers. Erythrocytic proteins from HHPA- and MHHPA-exposed workers were fractionated by gel filtration and ion exchange chromatography. In vitro synthesized conjugates between tritium-labeled and unlabeled HHPA and hemoglobin (Hb) were hydrolyzed by acid or digested by Pronase E. Levels of in vivo formed anhydride-Hb adducts and urinary/plasma levels of the corresponding acids were analyzed by gas chromatography-mass spectrometry (GC-MS) and correlated. The decay of adducts was studied in workers leaving employment or during vacation. More than 85% of the adduct forming protein in vivo coeluted with Hb in gel filtration and ion exchange chromatography. At least 70% of the HHPA in the in vitro formed adducts was found on lysine by GC-MS. Similar findings were obtained using Pronase E-digested tritium-labeled Hb-HHPA. The adduct levels in workers ranged 0-26 pmol/g Hb (mean 2. 7 pmol/g Hb) for HHPA, and the range for MHHPA was 0-55 pmol/g Hb (mean 4.1 pmol/g Hb). The Spearman's rank correlation coefficient between urine data and adducts was for HHPA rs = 0.80 and for MHHPA, rs = 0.78. For the plasma, the correlation using HHPA data was rs = 0.80 and for MHHPA, rs = 0.69. The adducts seemed to be stable in vivo. The adduct levels may be used as biomarkers of exposure to HHPA and MHHPA.

Determination of hexahydrophthalic acid and methylhexahydrophthalic acid in plasma after derivatisation with pentafluorobenzyl bromide using gas chromatography and mass spectrometric detection.

A method for the simultaneous determination of hexahydrophthalic acid (HHP acid) and methylhexahydrophthalic acid (MHHP acid) in human plasma was developed. The procedure was a rapid, single step extractive derivatisation with pentafluorobenzyl bromide as the derivatisation agent. The formed pentafluorobenzyl esters were analysed by gas chromatography-mass spectrometry in negative ion chemical ionisation mode with ammonia as the moderating gas. Deuterium-labeled HHP acid and MHHP acid were used as internal standards. The detection limit was 0.4 ng/ml for HHP acid (m/z 153) and 0.3 ng/ml for MHHP acid (m/z 365). The within-day precision of the method was between 2 and 3% and the between-day precision was between 3 and 12%. The overall recovery was between 65 and 83%. A comparison between HHP acid determinations with a previous and this method showed that the methods gave similar results. The method was applicable for analysis of plasma from occupationally exposed workers.

Biological monitoring of methylhexahydrophthalic anhydride by determination of methylhexahydrophthalic acid in urine and plasma from exposed workers.

OBJECTIVE: To investigate whether methylhexahydrophthalic acid (MHHP acid) in urine and plasma can be used as a biomarker for exposure to methylhexahydrophthalic anhydride (MHHPA). METHODS: MHHPA in air was sampled by Amberlite XAD-2 and analysed by gas chromatography (GC) with flame ionisation detection. MHHP acid in urine and plasma was analysed by GC with mass spectrometric detection. Workers occupationally exposed to MHHPA were studied. Air levels of MHHPA were determined by personal sampling in the breathing zone. Urinary levels of MHHP acid, a metabolite of MHHPA, were determined in 27 workers. In eight workers all urine was collected at intervals during 24 h. Plasma levels of MHHP acid were determined in 20 workers. RESULTS: The time-weighted average (TWA) air levels ranged from 5 to 60 micrograms MHHPA/m3 during 8-h workshifts. The urinary levels of MHHP acid increased during exposure and decayed after the end of exposure with an estimated half-life of about 6 h. A correlation was found between the TWA air levels of MHHPA and creatinine-adjusted MHHP acid levels in urine collected during the last 4 h of exposure. A correlation was also seen between the TWA air levels of MHHPA and the plasma concentrations of MHHP acid. An exposure to 20 micrograms MHHPA/m3 corresponded to about 140 nmol MHHP acid/mmol creatinine and about 40 nmol MHHP acid/l plasma. CONCLUSION: The results indicate that MHHP acid in urine or plasma may be used for biological monitoring of the exposure to MHHPA.

Direct measurement of hexahydrophthalic anhydride in workplace air with a transportable Fourier transform infrared spectrometer.

A method for direct measurement of hexahydrophthalic anhydride (HHPA) in workplace air by use of a Fourier transform infrared (FTIR) spectrometer was developed. Two visits were made to a plant manufacturing capacitors where HHPA was used. On the first visit a calibration method was developed according to what was expected to give the best calibration. This was performed by collection of 82 FTIR spectra from the air while simultaneously taking samples with a reference method using Amberlite XAD-2 sorbent tubes. On the second visit, two weeks later, the calibration method was used for prediction of HHPA concentrations (n = 52) in air; these were compared with XAD-2 determinations. The predicted FTIR values as a function of the XAD-2 determinations were used to evaluate some parameters regarding the FTIR method. The limit of detection was 120 micrograms HHPA/m3, and the precision at 150 micrograms/m3 was 22% and at 400 micrograms/m3 8%. When sampling from a pure HHPA atmosphere the obtained concentration by the FTIR was 103% of that of the XAD-2 tubes. The selection of different analytical parameters for the determinations are also discussed. The method is a useful tool in fast mappings of exposure levels.