Trimethylamine and trimethylamine oxide levels in normal women and women with bacterial vaginosis reflect a local metabolism in vaginal secretion as compared to urine.

The smell of rotten fish is one of the characteristics of bacterial vaginosis (BV), and is due to trimethylamine (TMA). Trimethylamine can be found in human urine, although most of it occurs as the nonvolatile oxide (TMAO) form. The fraction TMA/TMAO can be expected to be the same in different body fluids if no local production of TMA occurs. In women with BV, TMAO in the vaginal fluid is expected to be chemically reduced by the local bacterial flora to the much more odorous TMA. We have therefore studied the local vaginal production of TMA in vaginal secretion compared to the general TMA-TMAO metabolism that was measured in urine using gas chromatography. Both vaginal fluid and random urine samples were collected from women, with and without BV, attending a Swedish clinic for sexually transmitted diseases, and these samples were analyzed for TMA and TMAO. The results show that a local production of TMA occurs in the vagina that is not part of the general metabolism of TMA-TMAO.

PLUNC (palate, lung and nasal epithelial clone) proteins in human nasal lavage fluid.

PLUNC (palate, lung and nasal epithelial clone) is a newly discovered gene that is expressed in the upper respiratory tract and is suggested to be of importance in host defence against bacteria. We have identified two forms of the PLUNC protein in human nasal lavage fluid (NLF) using two-dimensional gel electrophoresis (2-DE) and MS. The apparent molecular masses and isoelectric points of these forms are 24.8 kDa/pI 5.4 and 25.1 kDa/pI 5.5. Notably, the 24.8 kDa/pI 5.4 form of PLUNC is an abundant protein in the 2-DE protein patterns of NLF from healthy subjects. Decreased levels of PLUNC were found in NLF from smokers and workers exposed to reactive epoxy chemicals, indicating that long-term exposure to airway irritants impairs the production of PLUNC in the upper respiratory tract. We have also investigated the presence of lipopolysaccharide (LPS)-binding proteins in NLF. Five proteins were found to adsorb to a LPS-coated surface; two of these proteins correspond to the two PLUNC forms, as judged by 2-DE pattern matching. For comparison, human saliva was found to contain a set of LPS-binding proteins with similar 2-DE spot positions (the same pIs but somewhat lower apparent molecular masses of approximately 20 kDa). These results indicate that PLUNC may be a new marker of airway inflammation and may play a part in the innate immune response, and that human saliva contains yet other members of the family of LPS-binding proteins.

Identification of a new potential airway irritation marker, palate lung nasal epithelial clone protein, in human nasal lavage fluid with two-dimensional electrophoresis and matrix-assisted laser desorption/ionization-time of flight.

We have analyzed protein patterns of human nasal lavage fluid (NLF) with two-dimensional gel electrophoresis (2-DE) and identified several proteins (such as transthyretin, Clara Cell protein 16, lipocalin-1, cystatin S, cystatin SN, immunoglobulin binding factor, statherin, calgranulin B, prolactin-inducible protein, and zinc-alpha2-glycoprotein) by N-terminal amino acid sequencing and matrix-assisted laser desorption/ionizationtime of flight (MALDI-TOF) mass spectrometry. To investigate whether airway irritation causes alterations in NLF 2-DE patterns, we compared epoxy workers with airway irritation (n=8) and healthy controls (n=6) before and after 2 h exposure to the epoxy chemical, dimethylbenzylamine (DMBA, 100 microg/m3) in an exposure chamber. A 25 kDa protein with pI 5.5 was found to be altered in the NLF 2-DE patterns; a trypsin digest of the 2-DE spot analyzed by MALDI-TOF and expressed sequence tags (ESTs) determined after post-source decay (PSD) identified the protein as palate lung and nasal epithelial clone (PLUNC). In controls, the levels of NLF-PLUNC were generally lower after 2 h exposure, whereas in epoxy workers, the levels were increased three- to twentyfold after exposure. The human gene sequence for PLUNC was just recently reported and so far no biofunctional data are available. Our results suggest that PLUNC is involved in the airway inflammatory response after exposure to irritants.

A prospective study of the relationship between exposure and specific antibodies in workers exposed to organic acid anhydrides.

BACKGROUND: The exposure-response relationships for the induction of specific IgE and IgG were evaluated in a prospective study of workers exposed to organic acid anhydrides (OAAs). Special attention was paid to the modifying effects of atopy and smoking. METHODS: The subjects were 163 previously unexposed persons exposed to epoxy resins with hexahydro-, methylhexahydro-, and methyltetrahydrophthalic anhydride as curing agents. The levels of OAAs in air and of specific IgE and IgG in serum were recurrently monitored. The mean observation time was 32 (1-105) months. RESULTS: The mean combined OAA exposure of the subjects was 15.4 (< 1-189) microg/m3. Positive specific IgE was demonstrated by 21 (13%) subjects with a mean induction time of 8.8 (1-35) months. The incidence of sensitization was 4.1 cases/1000 months at risk. The relative risk (OR) for atopics was 5.4 (1.9-15.3; 95% CI). An exposure-response relationship was demonstrated by an increasing risk of sensitization with increasing exposure. CONCLUSION: An association between exposure and atopy, respectively, and the induction of specific antibodies against OAAs were observed. The risk for atopics was comparable with the risk for the subjects in the most exposed group.

Newly identified proteins in human nasal and bronchoalveolar lavage fluids: potential biomedical and clinical applications.

Protein patterns of nasal lavage fluid (NLF) and bronchoalveolar lavage fluid (BALF) were analyzed with two-dimensional gel electrophoresis (2-DE) and a number of previously unidentified proteins (lipocalin-1, cystatin S, transthyretin, immunoglobulin binding factor and an 11 kDa fragment of albumin) were identified by N-terminal amino acid sequencing. Lipocalin-1 was shown to be a dominant protein in NLF from healthy subjects but was almost undetectable in NLF from a patient with asthma. It further appeared that lipocalin-1 in NLF consists of eight forms with pIs between 5.2 and 5.5: three with the expected Mr of 17500, two with increased Mr (18000), and three truncated variants with Mr of 17000. Two forms of cystatin S were identified both in NLF and BALF: one with pI 5.1 and Mr 13000, and the other with pI4.9 and Mr 13500. The distribution of the two forms was clearly different in NLF and BALF from healthy subjects with the 4.9/13500 form constituting only about 13% in NLF but 69% in BALF. In NLF from subjects with upper airway irritation a twofold increased proportion of the 4.9/13500 form was detected. Amino acid sequence data and the spot position indicate that the 4.9/13500 form might be a phosphorylated variant of cystatin S. Lower levels of both forms of cystatin S were found in BALF from smokers than nonsmokers. The levels of transthyretin in NLF were decreased in subjects exposed to irritating chemicals. Finally, higher levels of IgBF were found in BALF from smokers than nonsmokers. Taken together, these results illustrate the potential biomedical and clinical applications of identifying proteins in 2-DE patterns of human BALF and NLF. The possibility to describe and monitor airway disorders at the molecular level is inferred.

Toxicokinetics of 1,2,4-trimethylbenzene in humans exposed to vapours of white spirit: comparison with exposure to 1,2,4-trimethylbenzene alone.

This study compares the toxicokinetics of inhaled 1,2,4-trimethylbenzene (124TMB) in men exposed to white spirit with that previously observed in the same individuals exposed to 124TMB alone. The appropriateness of using dimethylhippuric acid (DMHA) metabolites of 124-, 123- and 135TMB in urine as biomarkers of exposure is also addressed and the kinetics of n-decane, n-undecane and 123TMB is investigated. The toxicokinetics of 124TMB was studied in nine male, healthy volunteers exposed to solvent vapours in an exposure chamber for 2 h during a work load of 50 W. The subjects were exposed to 2 ppm (11 mg/m3) of 124TM B during exposure to 300 mg/m3 of white spirit. The 124TMB isomer was analysed in blood, urine and exhaled air by gas chromatography. The DMHA metabolites of all three TMB isomers were analysed in urine by high-performance liquid chromatography. The results were compared with previously published exposures to 2 and 25 ppm (120 mg/m3) of 124TMB vapour alone. In addition, the occurrence of acute effects was studied by means of a questionnaire. Irritation and central nervous system (CNS) symptoms were recorded by ratings on a 100 mm visual analogue scale. Blood levels of 124TMB and excretion rates of 3,4-DMHA in urine were markedly elevated both during and after exposure to white spirit compared to the same exposure level of 124TMB alone. No irritation or CNS effects were reported in the questionnaire at any exposure condition. It appears that components in white spirit interfere with the metabolic elimination of 124TMB. This should be considered in biological exposure monitoring as well as in risk assessment.

Protein patterns of human nasal and bronchoalveolar lavage fluids analyzed with two-dimensional gel electrophoresis.

We have previously described the protein patterns of human nasal lavage fluid (NLF) and bronchoalveolar lavage fluid (BALF) after two-dimensional gel electrophoresis (2-DE). We now report the identification of a number of additional proteins in these 2-DE patterns. Several plasma proteins (alpha2-macroglobulin, haptoglobin alpha1-chain, IgA S chain, ceruloplasmin, alpha1-microglobulin, amyloid P and apolipoprotein A-1) could be included both in the BALF and NLF spot pattern data bases by matching with a master plasma 2-DE pattern (SWISS-2DPAGE). Furthermore, lysozyme, lactoferrin and the antiinflammatory proteins lipocortin-1 and Clara cell protein 16 (CC-16) were identified by matching with reference proteins and Western immunoblots. Significant differences in the levels of some of the identified proteins were found between NLF and BALF, and between BALF from smokers and nonsmokers. Transferrin, hemopexin and haptoglobin alpha1 were lower in NLF than BALF, while IgA, lysozyme and lactoferrin were higher in NLF than BALF. One form of alpha1-microglobulin was more abundant in NLF than in BALF, while the opposite was found for a second form of the same protein. Moreover, the levels of IgA, ceruloplasmin and the pro-form of apolipoprotein A-1 in BALF were lower in smokers than in nonsmokers. The possibility to describe and analyze differences in NLF and BALF 2-DE patterns at the protein spot level may have wide clinical applications.

Inhalation toxicokinetics of 1,2,4-trimethylbenzene in volunteers: comparison between exposure to white spirit and 1,2,4-trimethylbenzene alone.

The objective of this study was to compare the toxicokinetics of inhaled 1,2,4-trimethylbenzene (1,2,4-TMB) in man after exposure to white spirit with that observed after exposure to 1,2,4-TMB alone. TMBs occur mainly in petroleum products and the TMBs or their metabolites have been suggested as suitable biomarkers of exposure to white spirit and other distillation products. The toxicokinetics were studied in 9 male, healthy volunteers exposed to solvent vapours in an exposure chamber for 2 h during a work load of 50 W. The subjects were exposed to 11 mg/m3 of 1,2,4-TMB on two occasions; during exposure to 1,2,4-TMB vapour alone and during exposure to 300 mg/m3 of white spirit. The 1,2,4-TMB isomer was analyzed in blood and exhaled air by gas chromatography. In addition, a major urinary metabolite of 1,2,4-TMB, 3,4-dimethylhippuric acid (3,4-DMHA), was analyzed by high performance liquid chromatography. Further the occurrence of acute effects was studied by means of a questionnaire. Irritation and central nervous system symptoms were recorded by ratings on a 100-mm visual analogue scale. Blood levels of 1,2,4-TMB and excretion rates of 3,4-DMHA in urine were markedly elevated both during and after exposure to white spirit as compared to exposure to TMB alone. Thus, it appears that components in white spirit inhibit the metabolic elimination of 1,2,4-TMB. This should be considered in biological exposure monitoring as well as in risk assessment. No irritation or central nervous system effects were reported at these conditions.

Metachromatic cells and eosinophils in the nasal mucosa and N,N-dimethylbenzylamine exposure.

Six healthy non-atopic male volunteers participated in a dose-response study of N,N-dimethylbenzylamine (DMBA), which is a reactive chemical used in epoxy systems. The effects on the nasal mucosa after inhalation of 0, 20, 45, 80 and 120 microg/m3 were studied by means of symptom recordings, acoustic rhinometry, nasal lavages and nasal cytology processed for light microscopy of metachromatic cells (MC) and eosinophils (EOS). Although only minor symptoms were provoked, the numbers of MC and Eos tended to increase in a dose-response fashion after inhalation of the chemical. No signs of degranulation of the cells were found, as the levels of tryptase and eosinophil cationic protein in the nasal lavages remained low at all DMBA exposure levels. We therefore conclude that a reactive chemical such as DMBA can influence MC and Eos in the nasal mucosa even at low dose levels without causing significant clinical symptoms.

Determination of dimethylhippuric acid isomers in urine by high-performance liquid chromatography.

In order to analyse metabolites in urine after trimethylbenzene (TMB) exposure a method based on high-performance liquid chromatography (HPLC) for determination of the six dimethylhippuric acids (2,3-DMHA, 2,6-DMHA, 2,5-DMHA, 2,4-DMHA, 3,4-DMHA and 3,5-DMHA) in urine has been developed. In contrast to earlier published methods, the present method allows detection of all possible isomers of DMHA in a single analysis. The DMHAs were extracted from urine with dichloromethane. After evaporation, the residue was dissolved in mobile phase and analysed by a stepwise gradient HPLC system with ultraviolet (UV) detection at 225 nm. Mobile phase A (1.25% acetonitrile and 0.3% acetic acid in water) was used up to a retention time of 59.5 min and mobile phase B (5% acetonitrile in water containing 0.3% acetic acid) was used for completion of the analysis at approximately 90 min. The DMHA isomers were chromatographed on a reversed phase Radial-Pak C18 column (4 microns; 100 mm x 5 mm inner diameter). The detection limit for the six isomers was 1.5 micrograms/ml (range 0.5-3.4, 100 microliters injection volume). The precision of the method was 4.2% relative standard deviation (range 3.8-4.4; 100 micrograms/ml). Standard curves of the DMHAs were linear over the interval 10-500 micrograms/ml in human urine. Individual DMHAs or the sum of DMHA isomers may be used as biological indicators of occupational exposure to TMBs.

Experimental human exposure to N,N-dimethylbenzylamine: generation of a controlled atmosphere and biological monitoring.

The aim of the present study was to develop a method for generation of dimethylbenzylamine (DMBA) atmospheres in an exposure chamber and to investigate the possibility of using urinary DMBA metabolites for biological monitoring of exposure to DMBA. A DMBA atmosphere was generated by use of the gas-permeation principle. Six health male volunteers were exposed for 8 h to DMBA at air levels of 20, 45, and 80 microns/m3. Air levels of DMBA were analyzed by gas chromatography (GC). The total urinary amount of DMBA (U-SumDMBA; DMBA and metabolites that can be reduced to DMBA, e.g., DMBAO) was analyzed using GC-mass spectrometry (MS). The exposure chamber maintained very low (0-130 micrograms/m3) and steady concentrations for several weeks. DMBA uptake by inhalation was 76%. The amine was quickly distributed and biotransformed to nearly 100%. DMBA was eliminated in the urine with a half-time of 4.3 h. More than 50% was eliminated within 2 h of exposure. However, at all exposure levels the subjects continued to excrete DMBA the next morning. There was a significant correlation between the exposure to DMBA and the U-SumDMBA. Thus, U-SumDMBA may become an important biomarker for monitoring of industrial exposure to DMBA.

Two-dimensional gel electrophoresis of nasal and bronchoalveolar lavage fluids after occupational exposure.

Human nasal lavage fluids (NLFs), and bronchoalveolar lavage fluids (BALFs) were analyzed with two-dimensional gel electrophoresis (2-DE) and the protein patterns were evaluated with a computerized imaging 2-DE system. With silver staining about 1000 spots were detected in 10 microgram samples of NLF or BALF. Both BALF and NLF 2-DE patterns showed similarities to a reference plasma pattern and about 25 plasma proteins were identified in NLF as well as in BALF. Comparison showed that the levels of albumin and transferrin appeared to be slightly higher in BALF than NLF, while the levels of IgA, IgG and haptoglobin beta were higher in NLF than in BALF. In contrast to BALF and blood plasma, NLF contained large amounts of a cluster of acidic proteins (pI 4.5-5.5) with molecular masses of 15-30 kDa. Distinct alterations in the NLF 2-DE patterns were found in a worker who developed an asthmatic condition with bronchial hyperreactivity after exposure to organic acid anhydrides. After exposure, 14 protein spots were increased and one decreased by a factor of > 3 as compared to the levels before exposure and compared to healthy individuals. This is the first study indicating that 2-DE of NLF may be used to investigate early changes in airway protein patterns induced by occupational exposure to irritating chemicals.

N,N-dimethylbenzylamine: occupational exposure, analysis and biological monitoring.

Dimethylbenzylamine (DMBA) is used in the production of epoxy resins. The aims of this study were to assess occupational exposure to DMBA and evaluate the usefulness of monitoring the urinary excretion of DMBA and DMBA metabolites as indicators of exposure to DMBA. A sensitive gas chromatographic method for analysis of DMBA in air and in urine has been developed. The detection limit for DMBA in air for a 60-l air sample collected in 10 ml absorption solution was 2 micrograms/m3 and in charcoal tubes, 0.3 micrograms/m3. The detection limit for DMBA in urine was 0.02 mg/l. Ten male workers manufacturing epoxy resin were monitored during a full shift in the working environment and urine samples were collected at the end of exposure. The mean exposure and the highest DMBA concentration observed in air were 18 micrograms/m3 (time-weighted average; range 3-48 micrograms/m3) and 91 micrograms/m3, respectively. The DMBA concentrations in the urine samples were below the detection limit. After reduction of the urine samples the DMBA concentrations (U-SumDMBA) ranged from 0.02 to 0.22 mg/l. There was significant correlation between the exposure to DMBA and the U-SumDMBA. This observation suggests that the U-SumDMBA in urine samples collected at the end of a shift is a useful indicator of occupational exposure to DMBA.

Occupational exposure to hexahydrophthalic anhydride: air analysis, percutaneous absorption, and biological monitoring.

Urinary hexahydrophthalic acid (HHP acid) levels were determined in 20 workers occupationally exposed to hexahydrophthalic anhydride (HHPA) air levels of 11-220 micrograms/m3. The levels of HHP acid in urine increased rapidly during exposure and the decreases were also rapid after the end of exposure. The elimination half-time of HHP acid was 5 h, which was significantly longer than in experimentally exposed volunteers, possibly indicating distribution to more than one compartment. There was a close correlation between time-weighted average levels of HHPA in air and creatinine-adjusted levels of HHP acid in urine collected during the last 4 h of exposure (r = 0.90), indicating that determination of urinary HHP acid levels is suitable as a method for biological monitoring of HHPA exposure. An air level of 100 micrograms/m3 corresponded to a postshift urinary HHP acid level of ca. 900 nmol/mmol creatinine in subjects performing light work for 8 h. Percutaneous absorption of HHPA was studied by application of HHPA in petrolatum to the back skin of three volunteers. The excreted amounts of HHP acid in urine, as a fraction of the totally applied amount of HHPA, were within intervals of 1.4%-4.5%, 0.2%-1.3%, and 0%-0.4% respectively, indicating that the contribution from percutaneous absorption is of minor importance in a method for biological monitoring.

Dimethylethylamine in mould core manufacturing: exposure, metabolism, and biological monitoring.

The exposure and metabolism of dimethylethylamine (DMEA) was studied in 12 mould core makers in four different foundries using the Ashland cold box technique. The mean time weighted average (TWA) full work shift DMEA exposure concentration was 3.7 mg/m3. Inhaled DMEA was excreted into urine as the original amine and as its metabolite dimethylethylamine-N-oxide (DMEAO). This metabolite made up a median of 87 (range 18-93) % of the sum of DMEA and DMEAO concentrations excreted into the urine. Occupational exposure did not significantly increase the urinary excretion of dimethylamine or methylethylamine. The data indicate half lives after the end of exposure for DMEA in urine of 1.5 hours and DMEAO of three hours. The postshift summed concentration of DMEA and DMEAO in plasma and urine is a good indicator of the TWA concentration in air during the workday, and might thus be used for biological monitoring. An air concentration of 10 mg/m3 corresponds to a urinary excretion of the summed amount of DMEA and DMEAO of 135 mmol/mol creatinine.

Visual disturbances in man as a result of experimental and occupational exposure to dimethylethylamine.

Experimental exposure of four volunteers to 40-50 mg/m3 of dimethylethylamine (DMEA) for eight hours caused irritation of the mucous membrane of their eyes, subjective visual disturbances (haze), and slight oedema of the corneal epithelium. The thickness of the cornea showed a slight but consistent increase in all four subjects at these exposures and in two subjects exposed to 10 mg/m3. Concentrations of 80 and 160 mg/m3 for 15 minutes caused eye irritation but no visual disturbances or corneal oedema. Occupational exposure for eight hours to about 25 mg/m3 of DMEA (with peaks above 100 mg/m3) was also associated with eye irritation, haze, and corneal oedema. The divergence between our findings and other reports in which visual disturbances occurred at lower concentrations during occupational exposure may be due to peak concentrations.

Experimental study on the metabolism of dimethylethylamine in man.

Dimethylethylamine (DMEA) is an aliphatic tertiary amine, which is used as a catalyst in the mould core manufacturing. During 8 h, four healthy volunteers were exposed to four different DMEA air concentrations (10, 20, 40 and 50 mg/m3; 20 mg/m3, two subjects only). DMEA was biotransformed into dimethylethylamine N-oxide (DMEAO). On average, DMEAO, accounted for 90% of the combined amount of DMEA and DMEAO excreted into the urine. The half-lives of DMEA and DMEAO in plasma were 1.3 and 3.0 h, respectively. The urinary excretion of DMEA and DMEAO followed a two-phase pattern. The half-lives in the first phase were 1.5 h for DMEA and 2.5 h for DMEAO. In the second phase, which started about 9 h after the end of exposure, half-lives of 7 h for DMEA and 8 h for DMEAO were recorded. The combined concentration of DMEA and DMEAO, in both plasma and urine, showed an excellent correlation with the air concentration of DMEA. Thus, both urinary excretion and plasma concentration can be used for biological monitoring of exposure to DMEA. An 8-h exposure to 10 mg DMEA/m3 corresponds to a postexposure plasma concentration and 2-h postexposure urinary excretion of 4.9 mumol/l and 75 mmol/mol creatinine, respectively.

Metabolism of triethylamine in polyurethane foam manufacturing workers.

In 20 workers studied before, during, and after exposure to triethylamine (TEA) in a polyurethane-foam producing plant the amount of TEA and its metabolite triethylamine-N-oxide (TEAO) excreted in urine corresponded to an average of 80% of the inhaled amount. An average of 27% was TEAO, but with a pronounced interindividual variation. Older subjects excreted more than younger ones; less than 0.3% was excreted as diethylamine. The data indicate half-lives for TEA and TEAO excretion in urine of about 3 hr. The postshift level of TEA in urine and plasma are good indicators of the time-weighted average air level during the preceding work day, and might thus be used for biological monitoring. An air level of 10 mg/m3 (proposed occupational standard) corresponds to a urinary excretion of 65 mmol TEA/mol creatinine and a plasma level of 1.9 mumol/liter (biological exposure indices).