Comparison of non-linear and linear models for estimating haemoglobin adduct stability

According to the kinetic theory for the build-up and elimination of haemoglobin (Hb) adducts, unstable Hb adducts are simultaneously eliminated by zero-order Hb turnover and first-order chemical instability. Thus, the elimination of unstable Hb adducts is non-linear with respect to time. Nonetheless, many studies of Hb adduct stability have characterized the elimination of Hb adducts using linear zero-order or linear first-order models. This paper demonstrates the use of non-linear regression to estimate the first-order rate constant of Hb adduct instability (k) using data on the elimination of Hb adducts in rats dosed with benzene or ortho -toluidine. Results obtained using non-linear regression models are compared with results from the more commonly employed zero- and first-order linear models. It is shown that exposure estimates based on measured levels of unstable Hb adducts can be severely biased if zero-order turnover is assumed. Furthermore, based on published data, estimates of k are subject to estimated relative biases in the range of -4% to 96% when first-order linear models are used to characterize Hb adduct instability.     

Degree of alkylation of macromolecules in vivo from variable exposure.

Measurements of adducts formed with blood proteins, particularly haemoglobin, are increasingly being used to monitor human exposures to genotoxic chemicals. Information about the relationships between levels of genotoxic chemicals in the environment, e.g., concentration in the air, and levels of protein adducts in the blood is particularly important in setting safety standards and assessing risks. This paper describes the relationships between level of exposure to alkylating agents and level of haemoglobin adducts, considering the zero-order kinetics of the disappearance of these adducts. For comparison the corresponding relationship for adducts to macromolecules subjected to turnover, with first-order kinetics of disappearance, is described. For chemically stable and unstable adducts different exposure situations are considered: acute, chronic, intermittent and varying exposure levels. It is shown how an optimum solution of the problem of establishing the relationship between long-term exposure at varying levels (e.g., in work environments) and adduct level can be reached. Through mathematical derivations, which are given, expressions applicable to various exposure patterns are obtained and presented.     

Decay Studies of Dmpo-Spin Adducts of Free Radicals Produced by Reactions of Metmyoglobin and Methemoglobin with Hydrogen Peroxide

The 5, 5-dimethyl-1-pyrroline-N-oxide (DMPO) spin adduct of myoglobin (Mb) or hemoglobin (Hb) was formed when metmyoglobin (MetMb) or methemoglobin (MetHb) reacted with H₂O₂ in the presence of DMPO, and both decayed with half-life of a few minutes. The DMPO spin adduct of Mb decayed with biphasic kinetics with k₁ = 0.645 min^-1 and k₂ = 0.012 min^-1, indicating that the spin adduct consisted of two kinetically heterogeneous species, stable and unstable ones. The DPMO spin adduct of Hb, however, was homogeneous. Decay of both spin adducts was accelerated in the presence of tyrosine, tryptophan or cysteine, but not phenylalanine, methionine or histidine. The decay obeyed the first order kinetics at varying concentrations of the spin adducts. The decay was accelerated by denaturation and proteolysis of protein moiety. The decay rate was not affected by the extra addition of MetMb or MetHb to each spin adduct. The decay rate of the spin adduct of Mb was increased by hematin in the presence of H₂O₂ and decreased by catalase. Decay of stable spin adduct of Mb, however, was not significantly changed under any experimental conditions used. These results led us to conclude that instability of the DMPO-spin adducts of Mb and Hb is due to intramolecular redox reactions between the spin adducts and amino acid residues and/or products of the reaction between heme and H₂O₂.     

The research background for risk assessment of ethylene oxide: aspects of dose

Data for relationships between in vivo doses inferred from levels of hemoglobin (Hb) or DNA adducts and administered (by inhalation or injection) doses of ethylene oxide (EO) in mice, rats and humans are reviewed. At low absorbed doses or dose rates these relationships appear to be linear, whereas at higher dose rates deviations from linearity due to saturation kinetics of detoxification and of DNA repair as well as certain toxic effects have to be allowed for. If these factors are taken into consideration, a rather consistent picture is obtained for animal studies, with a variation by less than a factor 2 between estimates of adduct level increments or in vivo dose increments per unit of administered dose. Although the value for in vivo dose per unit of exposure dose (ppm-hour) in humans is uncertain because of unreliable data for the time-weighted average exposure level, the most likely value for this relationship, supported by data for ethene, agrees with data for the rodents. In the animal species testis doses are approximately one-half of the blood doses inferred from Hb adducts.     

Monitoring of occupational exposure to epichlorohydrin by genetic effects and hemoglobin adducts

The present work is focused on the determination of in vivo doses and studies of genetic effects in workers exposed to epichlorohydrin (ECH). The studied endpoints were hemoglobin (Hb) adducts, frequencies of hprt mutants, micronuclei in cytochalasin B blocked binucleated lymphocytes, sister chromatid exchanges (SCE) and high frequency cells (HFC). Blood samples were collected from office clerks and ECH exposed factory workers at an industrial plant in Germany. The workers were exposed to 0.11–0.23 ppm ECH in the air 45 h per week and to 0.2–2.6 ppm for 3 h per week. Some Swedish non-exposed subjects were also used for Hb adduct measurements. The genetic data, HFC and SCE, showed a significant difference between exposed and unexposed donors. In contrast to earlier studies on SCE, no impact of smoking was observed. Effects on micronuclei were on the borderline of significance, whereas there was no effect for HPRT mutants. The average Hb adduct level was higher in exposed than in non-exposed donors, although the difference was only significant when the exposed group was compared to Swedish controls. Smoking gave significantly increased adduct levels. The absence of significant correlations between individual data for Hb adducts and genetic effects, may be explained by the different periods of time covered by the responses in these endpoints. Whereas Hb adducts reflect the exposure during up to 4 months (i.e. the life span of human erythrocytes), the SCE, and particularly the HFC, seem to accumulate for years in a long-lived fraction of T-lymphocytes without DNA repair. Thus, the adduct data does not reflect the exposure backwards in time unless it can be shown that exposure conditions have remained unchanged. The origin of the background adduct levels in non-smoking control persons is at present not known.     

Benzo(a)pyrene diolepoxide adducts to albumin in workers exposed to polycyclic aromatic hydrocarbons: association with specific CYP1A1, GSTM1, GSTP1 and EHPX genotypes

We investigated whether the presence of (+)-anti-benzo(a)pyrene diolepoxide adducts to serum albumin (BPDE-SA) among workers exposed to benzo(a)pyrene (BaP) and unexposed reference controls was influenced by genetic polymorphisms of cytochrome P4501A1 (CYP1A1), microsomal epoxide hydrolase (EHPX), glutathione S-transferases M1 (GSTM1) and P1 (GSTP1), all involved in BaP metabolism. Exposed workers had significantly higher levels of adducts (0.124 ± 0.02 fmol BPTmg^-1 SA, mean ± SE) and a higher proportion of detectable adducts (40.3%) than controls (0.051 ± 0.01 fmol BPT mg^-1 SA; 16.1%) (p = 0:014 and p = 0:012). Smoking increased adduct levels only in occupationally exposed workers with the GSTM1 deletion (GSTM1 null) (p = 0:034).

Smokers from the exposed group had higher adduct levels when they were CYP1A1 *1/*1 wild-type rather than heterozygous and homozygous for the variant alleles (CYP1A1 *1/*2 plus *2/*2) (p = 0:01). The dependence of BPDE-SA adduct levels and frequency on the CYP1A1 *1/*1 genotype was most pronounced in GSTM1-deficient smokers. Exposed workers with GSTM1 null/GSTP1 variant alleles had fewer detectable adducts than those with the GSTM1 null/GSTP1*A wild-type allele, supporting for the first time the recent in vitro finding that GSTP1 variants may be more effective in the detoxification of BPDE than the wild-type allele. Logistic regression analysis indicated that occupational exposure, wild-type CYP1A1*1/*1 allele and the combination of GSTM1 null genotype+EHPX genotypes associated with predicted low enzyme activity were significant predictors of BPDE-SA adducts. Though our findings should be viewed with caution because of the relatively limited size of the population analysed, the interaction between these polymorphic enzymes and BPDE-SA adducts seems to be specific for high exposure and might have an impact on the quantitative risk estimates for exposure to polycyclic aromatic hydrocarbons.     

Hemoglobin adducts and urinary mercapturic acids in rats as biological indicators of butadiene exposure

Binding of 1,2-epoxy-3-butene, the primary metabolite of butadiene, to hemoglobin (Hb) and excretion of its mercapturic acid in urine were studies as potential indicators of butadiene exposure. Four groups of Wistar rats were exposed to butadiene at 0, 250, 500 and 1000 ppm 6 h/day, 5 days/week, during 2 weeks. Blood was collected at the end of exposure and 17 days later for analysis of hemoglobin adducts and adduct stability. Urine was collected each day during exposure (afternoon samples) and in between exposures (morning samples). Adducts of 1,2-epoxy-3-butene to N-terminal valine in Hb were measured using the N-alkyl Edman procedure and GC/MS of the thiohydantoin derivatives. The corresponding mercapturic acid was analysed, after deacetylation, through derivatization with phthaldialdehyde and HPLC with fluorescence detection. The Hb adducts proved to be stable and are therefore useful for dosimetry of long-term exposure to butadiene. The adduct levels increased linearly with exposure dose up to 1000 ppm (3 nmol/g Hb at 1000 ppm). The increase with exposure dose of the mercapturic acid concentration in urine was also compatible with a linear does response up to 1000 ppm. The sensitivity of both analytical methods needs to be improved for their application to human samples.     

Protein glycation, oxidation and nitration adduct residues and free adducts of cerebrospinal fluid in Alzheimer's disease and link to cognitive impairment

Increased damage to proteins by glycation, oxidation and nitration has been implicated in neuronal cell death leading to Alzheimer's disease (AD). Protein glycation, oxidation and nitration adducts are consequently formed. Quantitative screening of these adducts in CSF may provide a biochemical indicator for the diagnosis of AD. To assess this, we measured 11 glycation adducts, three oxidation adducts and a nitration adduct, determining both protein adduct residues and free adducts, in CSF samples of age-matched normal healthy subjects (n = 18) and subjects with Alzheimer's disease (n = 32). In CSF protein, the concentrations of 3-nitrotyrosine, N(epsilon)-carboxymethyl-lysine, 3-deoxyglucosone-derived hydroimidazolone and N-formylkynurenine residues were increased in subjects with Alzheimer's disease. In CSF ultrafiltrate, the concentrations of 3-nitrotyrosine, methylglyoxal-derived hydroimidazolone and glyoxal-derived hydroimidazolone free adducts were also increased. The Mini-Mental State Examination (MMSE) score correlated negatively with 3-nitrotyrosine residue concentration (p < 0.05), and the negative correlation with fructosyl-lysine residues just failed to reach significance (p = 0.052). Multiple linear regression gave a regression model of the MMSE score on 3-nitrotyrosine, fructosyl-lysine and N(epsilon)-carboxyethyl-lysine residues with p-values of 0.021, 0.031 and 0.052, respectively. These findings indicate that protein glycation, oxidation and nitration adduct residues and free adducts were increased in the CSF of subjects with Alzheimer's disease. A combination of nitration and glycation adduct estimates of CSF may provide an indicator for the diagnosis of Alzheimer's disease.     

The association between benzo[a]pyrene-DNA adducts and body mass index, calorie intake and physical activity

Prior work suggests that body size and fat content may influence carcinogen-DNA adduct levels measured in white blood cells. Here we consider energy balance more broadly by assessing the impact of body mass index (BMI), physical activity and calorie intake on the presence of benzo[a]pyrene-DNA (BP-DNA) adducts in white blood cell DNA. Our cross-sectional study employed subjects from a separately conducted intervention trial. Physical activity and food intake data were collected at 12 and 15 months of follow-up, respectively. BP-DNA adducts were measured by high-performance liquid chromatography (HPLC) in white blood cell samples collected at 12 months of follow-up. Complete data on all variables were available from 143 subjects. Logistic regression showed that BMI was inversely associated with the presence of detectable adducts (OR = 0.90, p=0.02), and that hours of moderate-intensity physical activity were positively associated with the presence of detectable adducts (OR = 1.04, p=0.04). These results provide further evidence that body fat content influences carcinogen-DNA adduct levels, probably by altering the distribution of the lipophilic parent compound.     

Computational methods for the screening of pharmacokinetic parameters in metabolism experiments

A computational strategy is presented which is useful for the qualitative and quantitative assessment of reaction-kinetic parameters in a one-compartment open model for the metabolism of substances in a biological system, especially the metabolism of dialkylnitrosamines to the ultimate carcinogen. The mathematical model used is that of first order kinetics and results in Bateman-functions. The strategy allows to decide about stability or instability of a substance occurring in a chain of transformations. In the case of stability quantitative estimates of the half life are provided. The procedure compares parameter estimates from models of different qualitative nature. These estimates are derived by linear or non-linear regression methods.     

Benzo(a)pyrene diolepoxide-haemoglobin and albumin adducts at low levels of benzo(a)pyrene exposure

A biomonitoring study was conducted to simultaneously measure individual benzo(a)pyrene (BaP) exposure in 50 office employees, not occupationally exposed to polycyclic aromatic hydrocarbons (PAH), using personal samplers and the formation of (+) r-7, t-8-dihyroxy-t-9,t-10-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (BPDE) adducts to haemoglobin (BPDE-Hb) and serum albumin (BPDE-SA). The population enrolled was exposed to an average of 0.58 ± 0.46 ng BaP m^-3 (mean ± SD). The concentration of BaP collected from smokers' samples was double that from non-smokers (P = 0.007). BPDE adducts to Hb and SA were quantified as BaP tetrols released from hydrolysis of macromolecules and measured by high-resolution gas chromatography-negative ion chemical ionization-mass spectrometry. BPDE-Hb adducts were detected in 16% of the population and BPDE-SA adducts in 28%. Smoking did not affect adduct formation. When BaP personal monitoring data were used as the criterion of exposure, no correlation was found with the presence and the levels of BPDE-Hb and BPDE-SA adducts. Undetected sources of PAH, such as the diet, might markedly alter the exposure profile depicted by individual air sampling and affect the frequency and levels of protein biomarkers. This is the first comparative analysis of BPDE-Hb and BPDE-SA adducts, providing reference values for these biomarkers in a general urban population. However it is difficult to establish which biomarkers would be the more relevant in assessing low BaP exposure, due to undetectable factors such as dietary PAHs, that might have influenced the results to some degree.     

DNA adducts as a biomarker of polycyclic aromatic hydrocarbon exposure in aquatic organisms: relationship to carcinogenicity

The use of DNA adduct measurement as a biomarker of exposure to polycyclic aromatic hydrocarbons (PAHs) is now well established in ecotoxicology. In particular, DNA adduct levels in aquatic organisms has been found to produce a better correlation with PAH exposure than PAH concentrations in organisms. DNA adducts levels are most commonly determined using the ³²P-postlabelling assay which measures total aromatic adducts. The relationship between relative DNA adduct formation and carcinogenicity has been investigated for a number of carcinogenic and non-carcinogenic PAHs using an in vitro system. Our results demonstrate that relatively high levels of DNA adducts can be produced by some non-carcinogenic PAHs, while other non-carcinogenic compounds do not produce detectable adducts. In addition, it has been shown that all carcinogenic PAHs investigated produce DNAadducts and that a relationship exists between relative adduct formation and carcinogenic potency. An investigation of adduct levels in fish liver and crustacean hepatopancreas in Oxley Ck, Brisbane has shown that higher than expected DNA adduct levels were correlated with the presence of carcinogenic and non-carcinogenic PAHs with high relative adduct forming potential.     

A new modified Edman procedure for analysis of N-terminal valine adducts in hemoglobin by LC–MS/MS

A rapid and sensitive method using liquid chromatography–tandem mass spectrometry (LC–MS/MS) for simultaneous determination of adducts from acrylamide, glycidamide and ethylene oxide to N-terminal valines in hemoglobin (Hb) was developed. This new procedure is based on the same principles as the N-alkyl Edman procedure for analysis of adducts from electrophilic agents to N-terminal valines in Hb. The N-substituted valines can be detached, enriched and measured selectively as thiohydantoins by the use of an Edman reagent, in this case fluorescein isothiocyanate (FITC). This procedure is denoted as the “adduct FIRE procedure” as the FITC reagent is used for measurement of adducts (R) formed from electrophilic compounds with a modified Edman procedure. In this study, fluorescein thiohydantoin (FTH) analytes of N-substituted valines from acrylamide, glycidamide and ethylene oxide, as well as their corresponding hepta- and tri-deuterium-substituted analogues, were synthesized. These analytes (n = 8) were then characterized by LC–MS/MS (ESI, positive ion mode) and obtained product ions were interpreted. A considerable work with optimization of the FIRE procedure™, resulted in a procedure in which low background levels of the studied adducts could be measured from 250 μL lyzed whole blood samples (human non-smokers). The analytes were enriched and purified with solid phase extraction columns and analyzed by LC–MS/MS with LOQ down to 1 pmol adduct/g Hb. Compared to other procedures for determination of N-terminal Hb adducts, the introduction of FITC has led to a simplified procedure, where whole blood also can be used, giving new opportunities and reduced hand on time with increased sample throughput.     

Combination of high-performance liquid chromatography and thin-layer chromatography separation of five adducted nucleotides isolated from liver resulting from intraperitoneal administration with 7H-dibenzo[c,g]carbazole to mice

7H-Dibenzo[c,g]carbazole, DBC, is a potent environmental liver carcinogen. Liver DNA from mice treated with DBC exhibited seven distinct DBC-DNA adducts as detected by ³²P-postlabeling using multidimensional TLC. To improve quantitation and chemically characterize the adducts, DNA samples were hydrlyzed, ³²P-postlabeled and the adducts were separated from the unadducted normal nucleotides on TLC using a D1 solvent, 0.65 M sodium phosphate (pH 6.8). Adducts were eluted from the TLC plates with 4.0 M pyridinium formate, concentrated, resuspended in 50% aqueous methanol and injected onto the HPLC; five individual adduct peaks were resolved and collected by this method. This approach will prove useful to decrease analysis time and improve chemical characterization of tightly clustered DNA adducts generated in vivo.     

Exposure-dependent accumulation of N-(2-hydroxypropyl)valine in hemoglobin of F344 rats exposed to propylene oxide by the inhalation route

The detection of hemoglobin adducts by mass spectrometry is a very sensitive and specific measurement of the extent of covalent binding of electrophilic chemicals. The exposure-dependent accumulation of N-(2-hydroxypropyl)valine (N-HPVal) in globin of rats exposed to propylene oxide (PO) (0, 5, 25, 50, 300 or 500 ppm) by the inhalation route was measured to assess the utility of Hb adducts as biomarkers of exposure. Analysis of N-HPVal by gas-chromatography tandem mass spectrometry showed a linear exposure-dependent response for adduct accumulation in globin of rats exposed to PO for 3 days (6 h/day). After 20 days of exposure (6 h/day; 5 days/week), the exposure-response curve was slightly sub-linear. DNA adducts had been measured in several organs of the same animals in a companion study. The dose-response for accumulation of DNA adducts was similar to that obtained for Hb adducts. However, the number of DNA adducts varied by 17-fold between different tissues. The highest number of DNA adducts was found in respiratory nasal tissue, followed by lung and then liver. These data demonstrate that hemoglobin adducts provide a sensitive dosimeter for systemic exposure, but cannot be used to predict the extent of DNA binding in individual tissues. Furthermore, the exposure-response curve for both hemoglobin and DNA adduct accumulation does not reflect the tumor incidence curve for PO, providing evidence that the assessment of risk to cancer is more complex than simple biomarker measurements. When the present rat data were compared with recent N-HPVal measurements in humans, similar binding was found.     

Development of a competitive immunoassay for the determination of N-(2-hydroxyethyl)valine adducts in human haemoglobin and its application in biological monitoring

Ethylene oxide (EO) is an important industrial compound and a directly acting mutagen. Human exposure to it can be monitored by the determination of haemoglobin (Hb) adducts. An immunoassay that quantifies the N-terminal adduct N-(2-hydroxyethyl)valine in whole blood was developed and its potential usefulness as a tool for biologically monitoring occupational exposure demonstrated. Analytical reliability was confirmed in a comparative study with gas chromatography-mass spectrometry (range 0.040-589 nmol g^-1 Hb, correlation coefficient 0.98, n=10). The assay was configured as a competitive enzyme-linked immunosorbent assay to facilitate the rapid throughput of samples. The assay uses a whole blood matrix and has a working range of 10-10 000 pmol N-(2-hydroxethyl)valine g^-1 Hb. The assay does not appear to be affected by structurally similar metabolites and has been used to determine adducts in human blood samples. The first results from potentially exposed workers indicate the assay might be a powerful tool for the routine occupational biomonitoring of EO exposure.     

Differential modification of hemoglobin chains by acetaldehyde

Acetaldehyde-hemoglobin adducts have been suggested as potential markers for alcohol consumption. These adducts were formed in vitro with [14C]acetaldehyde and separated into hemoglobin subunits by cation-exchange chromatography to examine the relative modification of the alpha- and beta-chains. The effect of varying concentrations of acetaldehyde on the relative amounts of polypeptide adducts and on the specific radioactivities of undissociated hemoglobin (Hb) following reaction with hydroxymercurybenzoate (HMB) was also studied. There were linear relationships (P less than 0.05) between increasing levels of [14C]acetaldehyde (0.0, 0.1, 0.2, 0.5 mM) and the radioactivities of the alpha- and one of the two beta-chain adducts (22, 25, 53 dpm/mg Hb and 151, 272, 626 dpm/mg Hb, respectively). Increases in radioactivities of a minor unidentified hemoglobin adduct fraction were also observed. The ratios of specific radioactivities of beta-to alpha-chain (8.8 +/- 1.2 SEM) did not vary with the concentrations of acetaldehyde. Although the amounts of undissociated hemoglobin following reaction with HMB did not increase with increasing concentrations of acetaldehyde, the significant increase of specific radioactivities of this fraction (152, 1967, and 6562 dpm/mg Hb for 0.1, 0.2, and 0.5 mM acetaldehyde, respectively) suggested possible crosslinks within the tetramer or dimer. The amino acid analysis of alpha- and beta-subunit adducts formed with 0.1 and 0.5 mM acetaldehyde showed that unreacted cysteine residues were more often detected at the higher acetaldehyde concentration consistent with the formation of cysteine adducts labile to acid hydrolysis or the shielding of cysteine residues in acetaldehyde-modified Hb against the subunit separation by HMB treatment. Thus acetaldehyde reacts differentially with the alpha- and beta-hemoglobin subunits and with the undissociated hemoglobin molecule.     

基于分位数回归的长白落叶松人工林最大密度线

基于东北地区378块固定样地和415块临时样地的调查数据和Reineke方程,利用线性分位数回归技术建立了不同分位点(τ=0.90、0.95、0.99)下的长白落叶松人工林最大林分密度与林木平均胸径的关系模型,选出拟合长白落叶松人工林最大密度线的最优模型. 利用人为选取最大的拟合数据,采用最小二乘(OLS)和最大似然(ML)回归同时建立最大密度线模型. 采用极值统计理论的广义Pareto模型推算现实林分特定径阶的极限最大株数,进一步建立极限密度线模型. 将线性分位数回归模型与其他方法进行对比.结果表明: 在全部径阶范围内选取5个最大数据点拟合的方法能够得到现实林分的最大密度线,选取的样点过多会使模拟结果偏离最大密度线,且ML法要优于OLS法. 分位点为0.99的线性分位数回归模型能够取得与ML接近的拟合结果,但分位数回归模型参数的估计结果更稳定. 人为选取拟合数据具有一定的人为性,最终选取分位点为0.99的分位数回归模型为拟合最大密度线的最优模型,参数估计结果为k=11.790、β=-1.586,极限密度线模型的参数估计结果为k=11.820、β=-1.594. 所确定的极限密度线位置略高于最大密度线,但二者差异不明显. 由固定样地数据的验证结果可知,所建立的最大林分密度线及极限密度线能够对现实林分的最大密度及极限密度进行预测,为长白落叶松人工林的合理经营提供依据.     

Mass spectral analyses of hemoglobin adducts formed after in vitro exposure of erythrocytes to hydroxymethylvinyl ketone

Hydroxymethylvinyl ketone (HMVK) is a reactive oxidation product of 3-butene-1,2-diol, a metabolite of 1,3-butadiene. The potential for HMVK (0.1 and 1mM) to form hemoglobin (Hb) adducts in erythrocytes from Sprague-Dawley rats was investigated at physiological conditions (pH 7.4, 37 degrees C) using electrospray ionization mass spectrometry (ESI/MS). With the 0.1mM HMVK globin samples, the results indicate HMVK adduction on the alpha2, beta2 and beta3 chains. With the 1.0mM HMVK globin samples, adducts were detected on the beta2 and beta3 chains. However, no correlation was observed between incubation time and the extent of adduct formation, and additional adducts were detected when globin samples were fractionated by HPLC before the ESI/MS analyses. For specific localizations of adducts on the globin chains, trypsin digested peptides from the 1mM HMVK globin samples were subjected to liquid chromatography/mass spectrometry analyses. The results, which are consistent with formation of HMVK adducts on several specific peptides within the alpha- and beta-chains, suggest selectivity in the interaction of HMVK with the different cysteine residues in Hb. Because adducts were also detected in peptides containing no cysteine residues and multiple HMVK moieties were detected on some of the cysteine-containing peptides, the results suggest other amino acids may be also reactive with HMVK. Adduct profiles and their relative intensities were consistent between the 1 and 2h samples providing evidence for the HMVK reactions being fast and selective. The finding that fewer peptides were adducted in the 0.1mM HMVK globin samples provides further evidence for selectivity of the HMVK reaction. Collectively, the results show HMVK readily and selectively forms adducts on Hb. Characterization of these adducts will facilitate development of useful biomarkers of exposure to HMVK and its precursor 1,3-butadiene.     

Merging Spline Approximations with Analysis of Nonlinear Regression Models

The paper outlines a method of analysis of intricically non-linear regression functions. The nonlinear model is first approximated by a cubic spline function. Thereafter, the standard methods of analysis in linear models are applied to obtain estimates and test statistics.