Plasma Protein Adducts Research Articles

Relationship between 4-Hydroxynonenal (4-HNE) as Systemic Biomarker of Lipid Peroxidation and Metabolomic Profiling of Patients with Prostate Cancer.

An oxidative degradation product of the polyunsaturated fatty acids, 4-hydroxynonenal (4-HNE), is of particular interest in cancer research due to its concentration-dependent pleiotropic activities affecting cellular antioxidants, metabolism, and growth control. Although an increase in oxidative stress and lipid peroxidation was already associated with prostate cancer progression a few decades ago, the knowledge of the involvement of 4-HNE in prostate cancer tumorigenesis is limited. This study investigated the appearance of 4-HNE-protein adducts in prostate cancer tissue by immunohistochemistry using a genuine 4-HNE monoclonal antibody. Plasma samples of the same patients and samples of the healthy controls were also analyzed for the presence of 4-HNE-protein adducts, followed by metabolic profiling using LC-ESI-QTOF-MS and GC-EI-Q-MS. Finally, the analysis of the metabolic pathways affected by 4-HNE was performed. The obtained results revealed the absence of 4-HNE-protein adducts in prostate carcinoma tissue but increased 4-HNE-protein levels in the plasma of these patients. Metabolomics revealed a positive association of different long-chain and medium-chain fatty acids with the presence of prostate cancer. Furthermore, while linoleic acid positively correlated with the levels of 4-HNE-protein adducts in the blood of healthy men, no correlation was obtained for cancer patients indicating altered lipid metabolism in this case. The metabolic pathway of unsaturated fatty acids biosynthesis emerged as significantly affected by 4-HNE. Overall, this is the first study linking 4-HNE adduction to plasma proteins with specific alterations in the plasma metabolome of prostate cancer patients. This study revealed that increased 4-HNE plasma protein adducts could modulate the unsaturated fatty acids biosynthesis pathway. It is yet to be determined if this is a direct result of 4-HNE or whether they are produced by the same underlying mechanisms. Further mechanistic studies are needed to grasp the biological significance of the observed changes in prostate cancer tumorigenesis.

Study of the Stability of Sulfur Mustard–Plasma Protein Adducts by Gas Chromatography–Tandem Mass Spectrometry

Study of the Stability of Sulfur Mustard–Plasma Protein Adducts by Gas Chromatography–Tandem Mass Spectrometry

Mass spectrometric analysis of adducts of sulfur mustard analogues to human plasma proteins: approach towards chemical provenancing in biomedical samples.

The primary aim of this study was to identify biomarkers of exposure to some so-called Schedule 1 sulfur mustard (HD) analogues, in order to facilitate and expedite their retrospective analysis in case of alleged use of such compounds. Since these HD analogues can be regarded as model compounds for possible impurities of HD formed during synthesis processes, the secondary aim was to explore to which extent these biomarkers can be used for chemical provenancing of HD in case biomedical samples are available. While the use of chemical attribution signatures (CAS) for neat chemicals or for environmental samples has been addressed quite frequently, the use of CAS for investigating impurities in biomedical samples has been addressed only scarcely. Human plasma was exposed to each of the five HD analogues. After pronase or proteinase K digestion of precipitated protein and sample work-up, the histidine (His) and tripeptide (CPF) adducts to proteins were analyzed, respectively. Adducts of the analogues could still be unambiguously identified next to the main HD adducts in processed plasma samples after exposure to HD mixed with each of the analogues, at a 1% level relative to HD. In conclusion, we have identified plasma protein adducts of a number of HD analogues, which can be used as biomarkers to assess an exposure to these Schedule 1 chemicals. We have shown that adducts of these analogues can still be analyzed after work-up of plasma samples which had been exposed to these analogues in a mixture with HD, supporting the hypothesis that biomedical sample analysis might be useful for chemical provenancing.

PECULIARITIES OF METABOLITES TOXICOKINETICS OF G-TYPE ORGANOPHOSPHORUS NERVE AGENTS IN BIO FLUIDS OF RATS SUBECTED TO ANTIDOTE THERAPY

The effect of the Pelixim antidote on the possibility to detect markers of G type nerve agents sarin and soman was studies in bio probes obtained in an acute in vivo experiment after exposure of rats to organophosphorus nerve agents in doses of 0.5LD50. It was found out that the intake of equitoxic doses of soman and sarin leads to a decrease of acetylcholinesterase erythrocyte membrane (AChE) activity for up to 7 days after exposure. The effect of Pelixim on the recovery of erythrocyte AChE activity is mostly pronounced a day after sarin poisoning. The fluoride regeneration of nerve agents from blood plasma protein adducts was possible for up to 7 days after soman poisoning without antidotal therapy and for 3 days after with antidotal therapy; in case of sarin intoxication, fluoride regeneration was possible for 3 days regardless of the use of antidotal therapy . The antidote strongly affected the excretion of hydrolytic metabolite of sarin О-isopropyl methylphosphonic acid (IMPA) and had no effect on the excretion of hydrolytic metabolite of soman О-pinacolyl methylphosphonic acid (PMPA). A day after poisoning and Pelixim injection, IMPA was detected in urine at a level of 15.3 ng/ml, whereas its level in the urine samples of animals in the absence of antidotal therapy was 55.0 ng/ml; 3 days after poisoning, IMPA was detected at a level of 4.9 ng/ml exclusively in the urine of animals subjected to antidotal therapy. The urine levels of PMPA in animals subjected and not subjected to antidotal therapy were respectively 44 and 53 ng/ml a day after poisoning and 12 and 14 ng/ml respectively 3 days after poisoning. Thus, the antidote impact on the excretion profile of hydrolytic metabolites is more significant for sarin than that of soman.

Effect of Irbesartan treatment on plasma and urinary markers of protein damage in patients with type 2 diabetes and microalbuminuria

The aim of this study was to assess the effect of the angiotensin II receptor blocker Irbesartan on protein damage by glycation, oxidation and nitration in patients with type 2 diabetes and microalbuminuria. In a double-masked randomised crossover trial of 52 hypertensive type 2 diabetic patients, antihypertensive treatment was replaced with bendroflumethiazide. After 2-months wash-out, patients were treated randomly with Irbesartan 300, 600, and 900 mg o.d., each dose for 2 months in a three-way crossover study. Glycation, oxidation and nitration adduct residues in plasma protein and related urinary free adducts were determined by stable isotopic dilution analysis liquid chromatography-tandem mass spectrometry. Treatment with Irbesartan decreased urinary excretion of advanced glycation endproducts (AGEs)--methylglyoxal- and glyoxal-derived hydroimidazolones, MG-H1 and G-H1. Urinary AGEs were decreased by 30-32%. In plasma protein, treatment with Irbesartan increased content of glycation adducts Nε-fructosyl-lysine, AGEs Nε-carboxymethyl-lysine, Nε-carboxyethyl-lysine and pentosidine, and also increased content of oxidation markers N-formylkynurenine and dityrosine. This was attributed to decreased clearance of plasma protein modified by Nε-fructosyl-lysine and oxidative markers through the glomerular filter tightened by Irbesartan treatment. Treatment of patients with type 2 diabetes with Irbesartan decreased urinary excretion of MG-H1, G-H1 and 3-NT, which may result from decreased exposure to these AGEs. This is likely achieved by blocking angiotensin II signalling and related down-regulation of glyoxalase 1 and may contribute to health benefits of Irbesartan therapy.

Increased levels of 4HNE-protein plasma adducts in Rett syndrome

Objective Rett syndrome (RTT) is a neurological disorder and a leading cause of mental retardation in females. It is caused by mutations in methyl-CpG-binding protein 2 (MeCP2) gene and more rarely in cyclin-dependent kinase-like 5 (CDKL5) and forkhead box protein G1 (FOXG1) genes. Increased oxidative stress (OS) has been documented in MeCP2-RTT patients. Here, we evaluated the levels of 4-hydroxynonenal plasma protein adducts (4HNE-PAs) in MeCP2-, CDKL5-, and FOXG1-RTT and in their clinical variants. Design and methods 4HNE-PAs were determined by Western blot in plasma from healthy subjects and RTT patients. Results 4HNE-PAs levels were increased in MeCP2- and CDKL5-related RTT but not in FOXG1-related RTT. Conclusion These results showed that OS is present in RTT clinical variants and could play a key role in RTT pathogenesis. Under the OS point of view FOXG1-related RTT appears to be distinct from the MeCP2/CDKL5, suggesting a distinct mechanism involved in its pathogenesis.

Frailty Syndrome is Associated with Altered Circulating Redox Balance and Increased Markers of Oxidative Stress

Frailty syndrome (FS) is a condition described in aging and characterized by physical vulnerability to stress and lack of physiological reserve. In this study we aim to define whether circulating oxidative stress correlates to frailty in terms of glutathione balance and oxidative protein damage. In 62 elderly outpatients, classified as frail patients according to Fried's criteria, evaluation of reduced glutathione (GSH), oxidized glutathione (GSSG), tumor necrosis factor-alpha, malonaldehyde-(MDA) and 4-hydroxy-2,3-nonenal-(HNE) protein plasma adducts were performed. A significant increase in the GSSG was observed in patients with FS when compared to non-frail. No difference was shown in the GSH amount, suggesting a glutathione oxidation more than impairment of the synthesis. TNF-alpha, MDA- and HNE-adducts, were significantly higher in FS as compared to non-frail patients. A logistic regression model correlating FS with redox balance showed a close relationship between glutathione ratio (OR=1.8, 95% CI=1.2-2.5) and MDA adducts (OR=2.8, 95% CI=1.6-4.7) to frailty. Our findings show an association between oxidative imbalance and Frailty Syndrome. GSSG/GSH ratio and plasma protein adducts strongly predict the frailty conditions and seem to be reliable and easily measurable markers in the context of the multidimensional analysis of elderly patients.

Analysis for Plasma Protein Biomarkers Following an Accidental Human Exposure to Sulfur Mustard

Following an accidental human exposure to a vesicating agent, plasma samples were analyzed for specific biomarkers of sulfur mustard. One individual suffered chemical burns over 6.5% of the body surface area and required hospitalization; the second individual developed a single, small blister. Plasma specimens from both individuals were examined using two different assays. The first assay targeted sulfur mustard adducts to cysteine-34 of albumin using affinity chromatography, enzyme digestion, and analysis of the alkylated peptide fragment using liquid chromatography-tandem mass spectrometry. The second assay targeted alkylation sites of glutamic and aspartic acids of plasma proteins. Following precipitation of plasma proteins, the sulfur mustard adducts were cleaved from the protein using base, derivatized, and analyzed using gas chromatography-mass spectrometry. Samples obtained over a 42-day period from the individual requiring hospitalization produced positive results for sulfur mustard adducts using both assays. Observed levels of the sulfur mustard biomarker decreased by approximately 75% between days 2 and 42 for both assays. Samples obtained over a six-day period from the individual with a single, small blister produced positive results for the albumin adduct assay. Observed levels were much lower than levels from the hospitalized patient. Blood samples from suspected human exposures to sulfur mustard have only rarely been made available for analysis by sensitive and specific laboratory assays. The data presented here add significantly to the small database of information that currently exists on human biomarkers of sulfur mustard exposure, linking a well-documented exposure event with levels of plasma protein adducts.

Accumulation of free adduct glycation, oxidation, and nitration products follows acute loss of renal function

Glycation, oxidation, and nitration of endogenous proteins occur spontaneously and these modifications are also present in foods. Increased levels of these chemical changes are associated with chronic renal failure; however, little is known about acute kidney failure. We measured these modifications of plasma protein and related free adducts in plasma following bilateral nephrectomy and bilateral ureteral obstruction. Advanced glycation end-product (AGE) residues of plasma protein were increased 3 h post-surgery, and thereafter slowly decreased in all groups, reflecting changes in plasma protein synthesis and transcapillary flow post-surgery. Ureteral ligation increased oxidation and nitration adduct residues. There were, however, marked increases in AGE, dityrosine, or 3-nitrotyrosine free adducts in both nephrectomized and ureter-ligated rats compared to rats that had undergone sham operations. There were lower modified adduct concentrations in the ureter-ligated compared to the nephrectomized rats, reflecting residual glomerular filtration and tubular removal. There was no increase in glycated, oxidized, and nitrated proteins. Glyoxal and methylglyoxal were also increased in both renal failure models. Our study shows that the acute loss of renal function and urinary excretion leads to the accumulation of AGE, oxidation, and nitration free adducts in the plasma.

Analysis of biomarkers in rats and dogs exposed to polymeric methylenediphenyl diisocyanate (pMDI) and its glutathione adduct

Hemoglobin adducts (Hb-MDX) of monomeric methylenediphenyl diisocyanate (MDI) are often interpreted as indirect evidence of hydrolysis of the diisocyanate moiety to the respective amine (diphenylmethane-4,4′-diamine, 4,4′-MDA) which constitutes the rationale of using this biomarker as an internal dosimeter of exposure to putatively formed MDA. In contrast, more recently published data suggest that following inhalation the high concentration of glutathione (GSH) present in lungs favor an adduct formation with GSH and/or peptides/proteins rather than hydrolysis. The focus of this study was to test this alternate hypothesis, viz. whether Hb-MDX can also be formed by the GSH bis-adduct of monomeric MDI. The synthesized mMDI-GSH bis-adduct was administered to rats by single intratracheal instillation. Additional groups were dosed by gavage and intraperitoneal injection. Biomarkers of exposure were determined in blood (plasma protein and hemoglobin adducts) and urine after harsh alkaline and acid hydrolysis, respectively. Data from previous single inhalation exposure studies with aerosols of MDI and 4,4′-MDA in rats served as reference. As to whether N-acetylation plays any modifying role to yield these mMDI-specific biomarkers was addressed in similarly head-only exposed dogs, a species with no appreciable N-acetylation capacity whereas rats are strong N-acetylators. The results obtained suggest that biomarkers in blood from controlled exposures above current workplace standards of mMDI appear not to be suitable for reliable assessments of past exposures. The biomarkers typically used to assess past exposures to MDI were also identified following exposure to the MDI-GSH bis-adduct. Their yield was low but quite similar for MDI aerosol and the MDI-GSH bis-adduct, whilst that of MDA was distinctively higher. The findings of this study are supportive of a conceptual pathway that the MDI-derived biomarkers of exposure are formed through MDI-GSH adducts rather than MDA. Data from dogs support the findings from rats and show that N-acetylation does not appear to be an essential modifying factor. It is concluded that the yield of MDI-related markers of exposure is relatively low and dependent on the exposure dose (and route). MDA originating from hydrolyzed serum protein or hemoglobin appear to be confounded by false-positive background levels which are surmised to be associated with the method of hydrolysis. The determination of urinary biomarkers might be a useful tool to identify recent exposures (by any route). Due methodological uncertainties associated with the harsh hydrolysis of biological specimens may be reduced substantially when using incremental pre- to post-shift changes rather than relying solely on absolute data.

The effects of partial thiamin deficiency and oxidative stress (i.e., glyoxal and methylglyoxal) on the levels of α-oxoaldehyde plasma protein adducts in Fischer 344 rats

We hypothesized that in marginal thiamin deficiency intracellular α-oxoaldehydes form macromolecular adducts that could possibly be genotoxic in colon cells; and that in the presence of oxidative stress these effects are augmented because of decreased detoxification of these aldehydes. We have demonstrated that reduced dietary thiamin in F344 rats decreased transketolase activity and increased α-oxoaldehyde adduct levels. The methylglyoxal protein adduct level was not affected by oral glyoxal or methylglyoxal in the animals receiving thiamin at the control levels but was markedly increased in the animals on a thiamin-reduced diet. These observations are consistent with our suggestion that the induction of aberrant crypt foci with marginally thiamin-deficient diets may be a consequence of the formation of methylglyoxal adducts.

Role of benoxaprofen and flunoxaprofen acyl glucuronides in covalent binding to rat plasma and liver proteins in vivo

Benoxaprofen (BNX) has been implicated in rare but serious hepatotoxicity which led to its withdrawal from the world market. Flunoxaprofen (FLX), a structural analog, appears to be less toxic. It has been postulated that the nonsteroidal antiinflammatory drugs associated toxicity may be related to covalent modification of proteins by their reactive acyl glucuronides, and the extent of covalent protein binding depends on both reactivity of the acyl glucuronide and the exposure to the reactive metabolite. The disposition of BNX and FLX in rats were compared upon intravenous administration of 20 mg/kg of BNX, FLX or their metabolites. Covalent binding of BNX and FLX to plasma and liver proteins were also determined, and an immunochemical approach was used to detect their hepatic targets. Similar concentrations of plasma protein adducts for BNX and FLX were detected even though the AUC of BNX-glucuronide (BNX-G) was almost twice that of FLX-glucuronide (FLX-G). Similar concentrations of liver protein adducts for BNX and FLX were also detected at 8 h, however, the hepatobiliary exposure of BNX-G was only 1/3rd that of FLX-G indicating that BNX-G was more reactive than FLX-G, which was in agreement with in vitro data. Proteins of 110 and 70 kDa were the major liver protein targets modified by covalent attachment of BNX and FLX. In conclusion, measuring covalent binding to tissue proteins in animals in addition to plasma adducts should be considered when evaluating and comparing carboxylic acid analogs that form reactive acyl glucuronides.

4-Hydroxynonenal is Markedly Higher in Patients on a Standard Long-term Home Parenteral Nutrition

Parenteral nutrition, a commonly used procedure in patients with gastrointestinal disorders, may lead with time to liver steatosis and fibrosis, whose pathogenesis has yet to be elucidated. Oxidative stress and particularly lipid peroxidation likely contribute to the expression of such hepatobiliary complications, by means of their recognized proinflammatory and profibrogenic effects. To evaluate the adequacy against oxidative insult of a standard micronutrient supplementation in patients under long term parenteral nutrition, a comprehensive patterns of redox indices has been determined on peripheral blood samples from forty one adults in comparison to fifty eight blood donors taken as controls. A sustained oxidative stress in peripheral blood of home parenteral patients was observed. Of the two lipid peroxidation markers found to be markedly increased, namely fluorescent plasma protein adducts with malondialdehyde and 4-hydroxynonenal, respectively, only the second was statistically correlated with all the antioxidant-related changes consistently detected in the patients, namely decreased plasma α-tocopherol and selenium intake and higher erythrocyte oxidized glutathione.Plasma level of 4-hydroxynonenal-protein adducts appears to be a reliable and easily measurable marker of oxidative status, particularly indicated to monitor the adequacy of dietary regimen during parenteral nutrition.

Development, validation and characterization of an analytical method for the quantification of hydrolysable urinary metabolites and plasma protein adducts of 2,4- and 2,6-toluene diisocyanate, 1,5-naphthalene diisocyanate and 4,4'-methylenediphenyl diisocyanate

Occupational exposure to diisocyanates within the plastic industry causes irritation and disorders in the airway. The aim of this study was to develop, validate and characterize a method for the determination of 2,4-toluenediamine (2,4-TDA), 2,6-toluenediamine (2,6-TDA), 1,5-diaminonaphthalene (1,5-NDA) and 4,4′-methylenedianiline (4,4′-MDA) in hydrolysed urine and plasma, and to study the correlation between the plasma and urinary levels of these potential biomarkers of 2,4-toluene diisocyanate (2,4-TDI), 2,6-toluene diisocyanate (2,6-TDI), 1,5-naphthalene diisocyanate (1,5-NDI) and 4,4′-methylenediphenyl diisocyanate (4,4′-MDI), respectively. Samples were hydrolysed with 0.3 M NaOH at 100°C for 24 h. The diamines were extracted, derivatized with pentafluoropropionic acid anhydride, and quantified by selected ion monitoring on gas chromatography-mass spectrometry. The repeatability and reproducibility of the method were 7-18% and 7-19%, respectively. Dialysis experiments showed that the metabolites of 2,4-TDI, 2,6-TDI, 1,5-NDI and 4,4′-MDI in plasma were exclusively protein adducts. No free diamines were found in urine, indicating that all diisocyanate-related metabolites were in a conjugated form. For each diisocyanate-related biomarker, there were strongly significant correlations (p<0.001) between individual levels of metabolites in plasma and urine, with Spearman's rank correlation coefficient (rs) values of 0.74-0.90. The methods presented here will be valuable for the development of biological monitoring methods for diisocyanates.

Critical analysis of biomonitoring endpoints for measuring exposure to polymeric diphenyl-methane-4,4'-diisocyanate (MDI) in rats: a comparison of markers of exposure and markers of effect.

The object of this study was to compare the relative sensitivity of markers of exposure and effects in the lung of rats exposed to polymeric diphenyl-methane-4,4'-diisocyanate (pMDI) aerosol. Rats were repeatedly exposed to 12.9 mg pMDI/m(3) (6 h/day, 5 days/week for 14 days; exposure was from days 0--17 followed by a post-exposure period to day 35). Markers of exposure were determined in bronchoalveolar lavage (BAL), blood (haemoglobin, plasma proteins), and urine on days 1, 4, 11, 18, 21, 28 and 35. Markers of effects were determined at the same time points and focused on changes in BAL constituents. In BAL, a maximum increase of total protein occurred following the first exposure and levelled off subsequently whilst BAL cell-related endpoints increased in a time-dependent manner. The kinetics of formation and elimination of adducts differed appreciably from one dosimeter to another. Whilst haemoglobin adducts were integrated by the cumulative exposures, the incremental yield of adduct formation appeared to be dependent on pulmonary as well as yet unknown erythrocyte-related factors. Plasma protein adducts attained a plateau after 1 week of exposure. MDI-related metabolite levels in urine did not show any time-dependent changes during the entire exposure period and declined rapidly during the post-exposure period. Thus, the kinetics of the fractional loading and clearance of pulmonary and extrapulmonary dosimeters did not parallel each other, nor was there a clear correlation with the markers of effects. In summary, it is concluded that biomonitoring is a powerful tool for the comparative dosimetry of well-defined exposure regimens. However, especially for irritant agents demonstrating portal-of-entry effects, markers related to 'total body burden' may not necessarily predict the absence or presence of local responses occurring within the target organ, namely the lung. In all compartments, dosimeters related to higher oligomers of MDI demonstrated low bioavailability, i.e. their recovery was appreciably lower than expected.

The Application of Biological Monitoring and Effects of Ethanol and Phenobarbital on Plasma Protein Adducts Formed in Rats Exposed to Benzidine

The Application of Biological Monitoring and Effects of Ethanol and Phenobarbital on Plasma Protein Adducts Formed in Rats Exposed to Benzidine

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

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. 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. 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. TPPA levels of HHPA and MHHPA are excellent biomarkers of long-term exposure to anhydrides.

4,4'-Methylenedianiline in hydrolysed serum and urine from a worker exposed to thermal degradation products of methylene diphenyl diisocyanate elastomers.

A 45-year-old mechanic employed in blowing hot air (350 degrees-600 degrees C) onto the surface of a polyurethane methylene diphenyl diisocyanate (MDI) conveyer belt developed dyspnoea, rhinoconjunctivitis and fever. The illness was suggestive of an MDI-associated illness, compatible with both immediate hypersensitivity and a complement-mediated immune-complex reaction. In his serum there were specific IgG and IgE antibodies against MDI and other isocyanates, and high values of circulating immune complexes. The patient's blood and urine samples were analysed for the presence of 4,4'-methylenedianiline (MDA) in hydrolysed urine and plasma. MDA was derivatized to amides using pentafluoropropionic acid anhydride (PFPA). Gas chromatography-mass spectrometry determinations were made monitoring the (M-20; M = molecular mass) fragments from the MDA-PFPA and the [2H2]MDA-PFPA derivative. The first urine sample was obtained 22 h and the last sample 114 h after start of exposure. The urine concentrations of MDA were corrected for creatinine. The half-time of MDA was 70-80 h. The first serum sample was obtained 19 h and the last sample 1967 days after the start of exposure. The half-time was 21 days, which suggests the presence of MDI/MDA plasma protein adducts in the exposed worker.

Albumin adducts, hemoglobin adducts and urinary metabolites in workers exposed to 4,4'-methylenediphenyl diisocyanate.

4,4'-Methylenediphenyl diisocyanate (MDI) is the most widely used isocyanate in the manufacture of polyurethanes. MDI has been implicated as one of the major causes of occupational asthma. Hydrolysis of MDI can yield 4,4'-methylenedianiline (MDA), which is a suspected human carcinogen. Thus the need to monitor occupational exposure to MDI is of great significance. The use of air monitors alone has been found to be insufficient and there is a need for sensitive markers of recent and long-term exposure. We obtained biological samples from a group of 20 workers exposed to MDI vapor during the manufacture of polyurethane products. The air levels of MDI in the factory were measured using personal, work room and work station monitors. In most cases the levels were below detection limits. The blood and urine samples were analyzed for the presence of adducts and metabolites using GC-MS methods. Urinary base-extractable metabolites were found above control levels in 15 of the 20 workers and ranged from 0.035 to 0.83 pmol MDA/ml. The level of the acetylated metabolite N'-acetyl-4,4'-methylenedianiline (AcMDA) ranged from 0.13 to 7.61 pmol/ml. The amount of MDA released after acid hydrolysis was on average 6.5 times higher than the amount of free MDA and AcMDA present in urine. MDA was detected as a hemoglobin (Hb) adduct in all of the 20 subjects. The level ranged from 70 to 710 fmol/g Hb. In one individual the Hb adduct of AcMDA was detected. This is the first time a Hb adduct of AcMDA has been detected after occupational exposure to MDI. This is a further piece of evidence for the biological availability of the suspected human carcinogen MDA from in vivo hydrolysis of MDI. Plasma albumin conjugates of MDI can cause the onset of respiratory disorders in both man and animal models. Thus we investigated the presence of plasma protein adducts. The plasma MDA levels ranged from 0.25 to 5.4 pmol/ml. Up to 120 fmol/mg were found to be covalently bound to albumin.

Enhanced covalent binding of tolmetin to proteins in humans after multiple dosing.

The in vivo stability of tolmetin-plasma protein adducts was characterized in six healthy human volunteers after a 400 mg single dose and after a multiple-dose regimen of 400 mg tolmetin every 12 hours for 10 days. Although the mean +/- SD maximum bound concentration was only 2.72 +/- 0.98 ng drug/mg protein after a single dose, it was almost an order of magnitude higher after multiple dosing. The protein adduct exhibited an average half-life of 4.8 +/- 0.9 days in contrast to the much shorter 5-hour half-lives for tolmetin and its glucuronide.