Maria Ospina

Assessment of the Relation between Biomarkers for Smoking and Biomarkers for Acrylamide Exposure in Humans

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 ≤10 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 ≤10 ng/mL. The intersubject variability was profoundly smaller in the group with PC values of ≤10 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 on HbAA and HbGA. (Cancer Epidemiol Biomarkers Prev 2007;16(11):2471–8)

Analysis of Four Human Microsporidian Isolates by MALDI-TOF Mass Spectrometry

Abstract Spores of four species of microsporidia isolated from humans were analyzed by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) and specific biomarkers were found for each. The microsporidia analyzed included three species, Encephalitozoon cuniculi, Encephalitozoon hellem, and Encephalitozoon intestinalis and the fourth organism is the recently described Brachiola algerae. Whole spores, spore shells, and soluble fractions were applied directly to the MALDI target without further purification steps. MALDI-TOF MS analysis of both whole spores and soluble fractions of the four isolates revealed a group of unique, characteristic, and reproducible spectral markers in the mass range of 2,000–8,000 Da. Statistical analysis of the averaged centroided masses uncovered two distinct sets of unique peptides or biomarkers, one originated from whole spores and the other from soluble fractions, that can differentiate the four microsporidian species studied. MALDI-TOF MS analysis of whole organisms is a rapid, sensitive, and specific option to characterize microsporidian isolates and has the potential for several applications in parasitology.

Pilot Study on the Impact of Potato Chips Consumption on Biomarkers of Acrylamide Exposure

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.

High-performance liquid chromatography method to analyze free and total urinary pyridinoline and deoxypyridinoline

The pyridinium cross-links pyridinoline (PYD) and deoxypyridinoline (DPD) are established markers of bone resorption measured in blood and urine and are used to investigate bone metabolism and manage bone diseases. Unfortunately, the currently observed interlaboratory variability caused by inconsistent assay calibration limits the optimal use of these markers. A high-performance liquid chromatography (HPLC)-based assay was developed using synthetic PYD and DPD as calibrators to analyze free and total PYD and DPD in urine. The spectroscopic characteristics of the synthetic calibrators were identical to those of calibrators isolated from bone. The mean intraassay variabilities of the HPLC method were 4.1 and 3.8%, respectively, for total DPD and PYD and 9.8 and 9.5%, respectively, for free DPD and PYD. The mean interassay variabilities were 9.1 and 8.2% for total DPD and PYD and 8.6 and 7.0% for free DPD and PYD, respectively. The mean recoveries were 98.1% for total DPD, 100.8% for total PYD, 98.6% for free DPD, and 94.9% for free PYD. The method exhibits a good correlation with a commercial immunoassay and with other HPLC assays currently used in hospital laboratories.

LC/MS/MS Method for the Analysis of Acrylamide and Glycidamide Hemoglobin Adducts

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.

Exposure to organophosphate flame retardant chemicals in the U.S. general population: Data from the 2013–2014 National Health and Nutrition Examination Survey

BACKGROUND Use of organophosphate flame retardants (OPFRs) including tris(1,3-dichloro-2-propyl) phosphate, triphenyl phosphate, tris(1-chloro-2-propyl) phosphate, and tris-2-chloroethyl phosphate, in consumer products is on the rise because of the recent phase out of polybrominated diphenyl ether (PBDE) flame retardants. Some of these chemicals are also used as plasticizers or lubricants in many consumer products. OBJECTIVES To assess human exposure to these chlorinated and non-chlorinated organophosphates, and non-PBDE brominated chemicals in a representative sample of the U.S. general population 6years and older from the 2013-2014 National Health and Nutrition Examination Survey (NHANES). METHODS We used solid-phase extraction coupled to isotope dilution high-performance liquid chromatography-tandem mass spectrometry after enzymatic hydrolysis of conjugates to analyze 2666 NHANES urine samples for nine biomarkers: diphenyl phosphate (DPHP), bis(1,3-dichloro-2-propyl) phosphate (BDCIPP), bis-(1-chloro-2-propyl) phosphate (BCIPP), bis-2-chloroethyl phosphate (BCEP), di-n-butyl phosphate (DNBP), di-p-cresylphosphate (DpCP), di-o-cresylphosphate (DoCP), dibenzyl phosphate (DBzP), and 2,3,4,5-tetrabromobenzoic acid (TBBA). We calculated the geometric mean (GM) and distribution percentiles for the urinary concentrations (both in micrograms per liter [μg/L] and in micrograms per gram of creatinine). We only calculated GMs for analytes with an overall weighted frequency of detection >60%. For those analytes, we also a) determined weighted Pearson correlations among the log10-transformed concentrations, and b) used regression models to evaluate associations of various demographic parameters with urinary concentrations of these biomarkers. RESULTS We detected BDCIPP and DPHP in approximately 92% of study participants, BCEP in 89%, DNBP in 81%, and BCIPP in 61%. By contrast, we detected the other biomarkers much less frequently: DpCP (13%), DoCP (0.1%), TBBA (5%), and did not detect DBzP in any of the participants. Concentration ranges were highest for DPHP (<0.16-193μg/L), BDCIPP (<0.11-169μg/L), and BCEP (<0.08-110μg/L). Regardless of race/ethnicity, 6-11year old children had significantly higher BCEP adjusted GMs than other age groups. Females had significantly higher DPHP and BDCIPP adjusted GM than males, and were more likely than males to have DPHP concentrations above the 95th percentile (odds ratio=3.61; 95% confidence interval, 2.01-6.48). CONCLUSIONS Our results confirm findings from previous studies suggesting human exposure to OPFRs, and demonstrate, for the first time, widespread exposure to several OPFRs among a representative sample of the U.S. general population 6years of age and older. The observed differences in concentrations of certain OPFRs biomarkers by race/ethnicity, in children compared to other age groups, and in females compared to males may reflect differences in lifestyle and exposure patterns. These NHANES data can be used to stablish a nationally representative baseline of exposures to OPFRs and when combined with future 2-year survey data, to evaluate exposure trends.

Comparison of questionnaire-based estimation of pesticide residue intake from fruits and vegetables with urinary concentrations of pesticide biomarkers

We developed a pesticide residue burden score (PRBS) based on a food frequency questionnaire and surveillance data on food pesticide residues to characterize dietary exposure over the past year. In the present study, we evaluated the association of the PRBS with urinary concentrations of pesticide biomarkers. Fruit and vegetable (FV) intake was classified as having high (PRBS≥4) or low (PRBS<4) pesticide residues for 90 men from the EARTH study. Two urine samples per man were analyzed for seven biomarkers of organophosphate and pyrethroid insecticides, and the herbicide 2,4-dichlorophenoxyacetic acid. We used generalized estimating equations to analyze the association of the PRBS with urinary concentrations of pesticide biomarkers. Urinary concentrations of pesticide biomarkers were positively related to high pesticide FV intake but inversely related to low pesticide FV intake. The molar sum of urinary concentrations of pesticide biomarkers was 21% (95% confidence interval (CI): 2%, 44%) higher for each one serving/day increase in high pesticide FV intake, and 10% (95% CI: 1%, 18%) lower for each one serving/day increase in low pesticide FV intake. Furthermore, intake of high pesticide FVs positively related to most individual urinary biomarkers. Our findings support the usefulness of the PRBS approach to characterize dietary exposure to select pesticides.

Developing a reference system for the IFCC standardization of HbA2

The importance of hemoglobin A2 (HbA2) as an indicator of the presence of β-thalassemia was established many years ago. However, clinical application of recommended HbA2 cut off values is often hampered due to poor equivalence of HbA2 results among methods and laboratories. Thus, the IFCC standardization program for HbA2 was initiated in 2004 with the goal of achieving a complete reference system for this measurand. HbA2 standardization efforts are still in progress, including the development of a higher-order HbA2 reference measurement procedure and the preparation of a certified reference material in collaboration with the IRMM. Here, we review the past, present and future of HbA2 standardization and describe the current status of HbA2 testing.

Characterization of N-terminal formaldehyde adducts to hemoglobin†

A procedure to prepare and purify adducts of formaldehyde (FA) to the N-terminus of peptides was developed. FA-VHLTPEEK and FA-VLSPADK were produced with purities >95% upon incubation of the peptides with FA in phosphate-buffered saline (PBS) at a pH level of 7.4. The peptides were purified by preparative liquid chromatography and were characterized by their retention times in liquid chromatography, their fragmentation patterns obtained by tandem mass spectrometry, and their accurate mass and nuclear magnetic resonance measurements. This is the first time an imidazolidone-type structure has been reported for FA adducts. The same peptides were identified in tryptic digests of human hemoglobin incubated with FA at physiological conditions and in human hemoglobin specimens. These peptides are suitable for use as calibrators for the quantitative assessment of internal exposure to FA.

Ultraperformance Liquid Chromatography Tandem Mass Spectrometry Method To Determine Formaldehyde Hemoglobin Adducts in Humans as Biomarker for Formaldehyde Exposure

Formaldehyde (FA) is an environmental chemical classified as a human carcinogen. It is highly reactive and can bind covalently with hemoglobin (Hb) to produce Hb adducts. Measurement of these Hb adducts provides valuable information about exposure to this chemical. We developed a robust, ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for quantifying FA-Hb adducts in red blood cells. The method measures the FA-VHLTPEEK peptide after trypic digestion. The peptide is a FA adduct at the N-terminus of the beta chain of human Hb. Method mean (±SD) accuracy, determined by recovery in quality control and blank material was 103.2% ± 8.11. The mean among-day and within-day coefficients of variation determined at three concentration levels (%CV) were 9.2% (range: 7.2-10.2%) and 4.9% (range 3.1-7.3%), respectively. The limit of detection was 3.4 nmol/g Hb. This method was applied to the analysis of 135 human blood samples, and FA-VHLTPEEK was detected in all study samples. FA-VHLTPEEK concentrations were not significantly different between smokers and nonsmokers. This work is the first validated UPLC-MS/MS method in which a FA peptide derived from a FA-Hb adduct could be used to monitor exposure to FA in population studies.