Shewanella putrefaciens is a facultatively anaerobic bacterium in the gamma group of the proteobacteria, capable of utilizing a wide variety of anaerobic electron acceptors. An examination of its cytochrome content revealed the presence of a tetraheme, low-redox-potential (E 0 ‫؍‬ ؊233 mV), cytochrome c-type cytochrome with a molecular mass of 12,120 Da and a pI of 5.8. The electron spin resonance data indicate a bis-histidine coordination of heme groups. Reduction of ferric citrate was accompanied by oxidation of the cytochrome. The biochemical properties suggested that this protein was in the cytochrome c 3 group, which is supported by N-terminal sequence data up to the first heme binding site.The ability of Shewanella putrefaciens to grow with a variety of different electron acceptors including oxygen, nitrate, sulfur, thiosulfate, sulfite, fumarate, trimethylamine-N-oxide (TMAO), and metal ions (10, 11) raises questions about the architecture of the electron transport chain in this versatile organism. In particular, given the range of electron potentials covered by the various electron acceptors it utilizes, the question of which types and the numbers of cytochromes present becomes an interesting one. In this regard, little detailed work has been done on cytochromes from S. putrefaciens. Early work by Obuekwe and Westlake (12) with whole-cell spectra showed that cytochrome c-type cytochromes were present and that both the red cell coloration and cytochrome content were correlated with the presence of iron in the growth medium. Arnold et al. (2) also reported whole-cell spectra and concluded that cytochrome synthesis was induced by growth under conditions of low oxygen tension. Myers and Myers (9) studied the locations of cytochromes in S. putrefaciens, concluding that many c-type cytochromes were present and that they were primarily localized in the outer membrane. Morris et al. (7) resolved nine c-type cytochrome bands by DEAE-Sepharose chromatography. There were two principal components that eluted at 90 mM and 315 mM NaCl, both of which had low redox potentials. In all of the above-cited studies, cytochrome content and type were judged mainly from spectral data, using the intensity of the Soret band after treatment with dithionite, or by heme staining polyacrylamide gels with tetramethylbenzidine (18). In recent and more definitive studies, a large tetraheme flavocytochrome c fumarate reductase was purified from S. putrefaciens (NCIMB400), and the gene sequence was determined (6,13). This enzyme is a 63.8-kDa soluble protein, and unlike the usual membrane-bound fumarate reductase, it contains heme c instead of iron-sulfur centers, and the heme midpoint redox potentials of Ϫ220 and Ϫ320 mV are unusually low. In a recent study, Lies et al. (5) reported that S. putrefaciens produces isoprenoid quinones of three types, with menaquinones and isoprenoid types predominating under aerobic conditions and methylmenaquinone types being present under anaerobic growth conditions. In this study, we report the purificat...

Smoking is an important source of acrylamide exposure in the general population. We assessed the relationship between hemoglobin adducts of acrylamide (HbAA) and glycidamide (HbGA) as biomarkers of acrylamide exposure and plasma cotinine (PC) as biomarkers of tobacco smoke exposure in 94 men and 67 women. The median (5th-95th percentile) biomarker concentrations (pmol/g Hb) in the group of individuals with PC concentrations of V10 ng/mL were 51 (29-155) and 34 (16-117) for HbAA and HbGA, respectively. They were significantly lower than those in the group of individuals with PC concentrations of >10 ng/mL [194 (87-403) and 107 (41-215) for HbAA and HbGA, respectively]. In individuals with PC concentrations of <1 ng/mL, HbAA and HbGA were similar to those observed in the group with PC values of V10 ng/mL. The intersubject variability was profoundly smaller in the group with PC values of V10 ng/mL compared with the group with PC values of >10 ng/mL. Although HbAA and HbGA could be categorized into distinguishable groups using PC concentration ranges commonly used to categorize presumed smokers and nonsmokers, no significant relationship was observed between these two biomarkers and PC within each group. The different exposure periods reflected by these biomarkers and the resulting different susceptibility to short-term variations in exposure patterns may in part explain these observations. The findings suggest that tobacco smoke exposure in individuals with PC values of <1 ng/mL has only a minimal effect

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BackgroundThe lifelong exposure of the population to acrylamide has raised concerns about the possible health effects of the chemical. Data on the extent of exposure to acrylamide and its primary metabolite, glycidamide, are needed to aid in the assessment of potential health effects.ObjectivesThe aim of this study was to assess human exposure to acrylamide and glycidamide in the general U.S. population through the measurement of hemoglobin adducts of acrylamide (HbAA) and glycidamide (HbGA).MethodsHbAA and HbGA were measured in 7,166 subjects from the National Health and Nutrition Examination Survey. Stratified HbAA and HbGA data were reported by sex, age groups, race/ethnicity (Mexican American, non-Hispanic black, non-Hispanic white), and smoking status based on serum cotinine levels. Covariate-adjusted geometric means for each demographic group were calculated using multiple regression analysis.ResultsHbAA and HbGA levels ranged from 3 to 910 and from 4 to 756 pmol/g hemoglobin, respectively, with smokers having the highest levels overall. Tobacco smoke exposure in nonsmokers had a small but significant effect on HbAA and HbGA levels. Adjusted geometric mean levels for children 3–11 years of age were higher than for adults ≥ 60 years of age [mean (95% confidence interval): HbAA, 54.5 (49.1–51.5) and HbGA, 73.9 (71.3–76.6) vs. HbAA, 46.2 (44.3–48.2) and HbGA, 41.8 (38.7–45.2)]. Levels were highest in Mexican Americans [HbAA: 54.8 (51.9–57.8), HbGA: 57.9 (53.7–62.5)], whereas non-Hispanic blacks had the lowest HbGA levels [43.5 (41.1–45.9)].ConclusionsU.S. population levels of acrylamide and glycidamide adducts are described. The high variability among individuals but modest differences between population subgroups suggest that sex, age, and race/ethnicity do not strongly affect acrylamide exposure. Adduct concentration data can be used to estimate relative exposure and to validate intake estimates.

The general population is exposed to acrylamide, a potential human carcinogen, through food and cigarette smoke. The assessment of human exposure to acrylamide is important in the evaluation of health risks associated with this chemical. Hemoglobin adducts of acrylamide (AA-Hb) and its primary metabolite glycidamide (GA-Hb) are established biomarkers of acrylamide exposure and methods to measure these biomarkers using modified Edman reaction are described. Only limited information about the optimal Edman reaction conditions such as pH or temperature is available for these adducts and the existing methods do not allow automation needed in biomonitoring studies. In this study, the yield of Edman products of AA-Hb and GA-Hb between pH 3-10 and at 35-55 degrees C at different time intervals, and the applicability of liquid-liquid extraction on diatomaceous earth for analyte extraction, were assessed and results were used in a new optimized method. The applicability of our optimized method was assessed by comparing results obtained with a convenience sample from 96 individuals with a conventional method. Maximum yield of Edman products was obtained between pH 6-7, heating the reaction solution at 55 degrees C for 2 h resulted in the same yields as with conventional conditions, and use of diatomaceous earth was found suitable for automated analyte extraction. Using these conditions, no difference was observed between our optimized and a conventional method. The median globin adduct values in the convenience sample are 129 pmol/g globin (range: 27-453 pmol/g globin) and 97 pmol/g globin (range: 27-240 pmol/g globin) for AA-Hb and GA-Hb, respectively. The GA-Hb/AA-Hb ratio decreases significantly with increasing AA-Hb values indicating that measurement of AA-Hb as well as GA-Hb are needed to appropriately assess human exposure to acrylamide.

Food is assumed to be one major source of acrylamide exposure in the general population. Acrylamide exposure is usually assessed by measuring hemoglobin adducts of acrylamide and its primary metabolite glycidamide as biomarkers. Little is known about the impact of acrylamide in food on biomarkers of acrylamide exposure. Therefore, CDC is conducting a feeding study to investigate the effect of consumption of endogenous acrylamide in food on biomarkers of acrylamide exposure. As part of this study, we performed a pilot study to obtain further information on the magnitude of the changes in biomarker levels after consumption of high amounts of potato chips (21 ounces) over a short period of time (1 week) in non-smokers. After 1 week, biomarkers levels increased up to 46% for acrylamide adducts and 79% for glycidamide adducts. The results indicate that changes in biomarker levels due to consumption of potato chips can be detected. However, because of the design of this pilot study, the observed magnitude of change cannot be. generalized and needs to be confirmed in the main study.

Hemoglobin adducts of acrylamide and its primary metabolite, glycidamide are used as biomarkers of acrylamide exposure. Several methods for analyzing these biomarkers in blood have been described previously. These methods were developed to analyze small numbers of samples, not the high sample throughput that is needed in population screening. Obtaining data on exposure of the US population to acrylamide through food and other sources is important to initiate appropriate public health activities. As part of the Centers for Disease Control and Prevention biomonitoring activities, we developed a high throughput liquid chromatography tandem mass spectrometry (LC/MS/MS) method for hemoglobin adducts of acrylamide. The LC/MS/MS method consists of using the Edman reaction and isolating the reaction products by protein precipitation and solid-phase extraction (SPE). Quantitation is achieved by using stable-isotope labeled peptides as internal standards. The method is performed on an automated liquid handling and SPE system. It provides good sensitivity in the low-exposure range as assessed in pooled samples and enables differentiation between smokers and non smokers.

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