Article Cite This: Chem. Res. Toxicol. 2018, 31, 1240−1247
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Age-Associated Methylation in Human Hemoglobin and Its Stability on Dried Blood Spots As Analyzed by Nanoflow Liquid Chromatography Tandem Mass Spectrometry Hauh-Jyun Candy Chen* and Sun Wai Ip Department of Chemistry and Biochemistry, National Chung Cheng University, 168 University Road, Ming-Hsiung, Chia-Yi 62142, Taiwan
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ABSTRACT: Methylation of biomolecules is involved in many important biological processes. The contributing methylating agents arise from endogenous and exogenous sources (such as cigarette smoking). Human hemoglobin is easily accessible from blood and has been used as a molecular dosimeter for monitoring chemical exposure. We recently developed a method for characterization and quantification of the extents of methylation and ethylation in hemoglobin by nanoflow liquid chromatography tandem mass spectrometry under the selected reaction monitoring mode. Using this method, the relative extents of methylated and ethylated peptides in hemoglobin were quantified in nonsmoking subjects at various ages in this study. Among the nine methylation sites, we found that the extents of methylation were significantly higher in elderly subjects at the N-terminal and His-20 of α-globin, and at the Nterminal and Glu-26 of β-globin. Moreover, the extents of methylation at these sites were significantly correlated with the age of the subjects. On the other hand, no statistically significant difference was found in the ethylated peptides. We also examined the stability of methylated and ethylated hemoglobin when stored on dried blood spot cards. The extents of these modifications on hemoglobin are stable for at least 4 weeks stored at room temperature. Our results suggest that age should be considered as a factor when measuring hemoglobin methylation and that dried blood spot is a valuable biomonitoring technique for hemoglobin modifications in epidemiological studies.
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signal transduction, and so forth.7−10 S-Adenosylmethionine (SAM) is the major endogenous methyl donor for many endogenous processes11 and its high concentration in erythrocytes12 suggests that it may in part contribute to methylation of hemoglobin. In addition, humans are exposed to exogenous methylating agents that are known to methylate DNA and proteins.13−16 Exogenous methyl donors include those from the environment and cigarette smoke, such as the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N-nitrosodimethylamine.17 In our earlier report, the effect of cigarette smoking on the extent of methylation and ethylation at certain sites of hemoglobin was investigated.18 The results showed that the extent of ethylation at six sites of hemoglobin was significantly higher in smokers than in nonsmokers and correlated with the number of cigarettes smoked per day. On the other hand, no significant correlation was found between smoking and the methylated peptides in hemoglobin,18 which is in agreement with reports by the research groups of Törnqvist and Hecht.11,19,20 During the course of that study, we realized that age seemed to have certain effect on the extent of
INTRODUCTION Humans are constantly exposed to chemically reactive compounds, and the extent of exposure in tissues can be monitored through their reaction products (adducts) with biomolecules, such as DNA and proteins. For the purpose of biomonitoring, blood proteins have been preferred to DNA due to its high abundance and well-defined life span.1 We focus on hemoglobin adducts because human hemoglobin is abundant, easily obtainable, minimally invasive, and relatively long-lived (∼120 days),1 which provides an opportunity for the adducts to accumulate without being repaired. Although DNA damage is directly implicated in the carcinogenesis processes, levels of DNA adducts are much lower than those of protein adducts.1 This might be partly due to the fact that DNA is in the nucleus of a cell and protected by histone proteins. Most importantly, cells have developed sophisticated DNA repair mechanisms. In addition, linear dose−response curves are documented for both DNA and Hb adducts in animals and in humans exposed to the same carcinogens or reactive metabolites.2−4 Therefore, hemoglobin adducts can potentially serve as a surrogate for DNA adducts in molecular dosimetry to monitor chemical exposure.5,6 Methylation plays a central role in many important biological processes including regulation of transcription, © 2018 American Chemical Society
Received: August 14, 2018 Published: October 26, 2018 1240
DOI: 10.1021/acs.chemrestox.8b00224 Chem. Res. Toxicol. 2018, 31, 1240−1247
Article
Chemical Research in Toxicology
Table 1. Relative Extent of Methylation and Ethylation in Hemoglobin Isolated from Nonsmoking Subjects and Statistical Analysis relative extent of modification (mean ± SD) elder nonsmokers (n = 20)
Mann−Whitney U-test
young nonsmokers (n = 14)
α-1VMe α-20HMe α-50HMe α-72HMe β-1VMe β-26EMe β-66KMe β-77HMe β-93CMe
6.95 1.87 2.58 7.99 2.12 3.44 1.03 1.85 1.72
× × × × × × × × ×
10−04 10−03 10−04 10−05 10−04 10−02 10−04 10−03 10−04
± ± ± ± ± ± ± ± ±
6.66 1.02 2.58 1.19 6.55 1.93 4.80 2.08 7.08
× × × × × × × × ×
10−04 10−03 10−04 10−04 10−05 10−02 10−05 10−03 10−05
2.66 1.19 2.86 3.54 9.74 1.97 1.20 9.30 1.72
× × × × × × × × ×
10−04 10−03 10−04 10−05 10−05 10−02 10−04 10−04 10−04
± ± ± ± ± ± ± ± ±
8.97 6.95 1.14 5.07 5.06 1.31 1.17 1.88 9.14
× × × × × × × × ×
10−05 10−04 10−04 10−05 10−05 10−02 10−04 10−04 10−05
α-1VEt α-16KEt α-50HEt α-72HEt α-87HEt β-1VEt β-17KEt β-66KEt β-77HEt β-92HEt β-93CEt
8.35 6.25 1.64 3.40 2.55 2.11 3.60 1.20 4.08 3.64 8.82
× × × × × × × × × × ×
10−05 10−05 10−05 10−06 10−05 10−05 10−05 10−05 10−06 10−05 10−05
± ± ± ± ± ± ± ± ± ± ±
8.61 5.05 1.38 4.24 1.88 1.44 6.19 1.30 2.74 3.27 1.40
× × × × × × × × × × ×
10−05 10−05 10−05 10−06 10−05 10−05 10−05 10−05 10−06 10−05 10−04
1.07 1.03 3.79 3.91 3.18 2.05 1.01 2.64 4.16 3.17 1.25
× × × × × × × × × × ×
10−04 10−04 10−05 10−06 10−05 10−05 10−04 10−05 10−06 10−05 10−04
± ± ± ± ± ± ± ± ± ± ±
1.04 7.42 4.96 4.65 2.11 1.53 9.85 2.70 3.17 2.08 2.56
× × × × × × × × × × ×
10−04 10−05 10−05 10−06 10−05 10−05 10−05 10−05 10−06 10−05 10−04
p value
a
Spearman correlation coefficient (p value)b age
0.0082 0.0263
0.4640 (0.0057) 0.3601 (0.0365)
