Correlation between Glyoxal-Induced DNA Cross-Links and Hemoglobin Modifications in Human Blood Measured by Mass Spectrometry

Abstract

Glyoxal is an oxoaldehyde generated from the degradation of glucose-protein conjugates and from lipid peroxidation in foods and in vivo, and it is also present in the environment (e.g., cigarette smoke). The major endogenous source of glyoxal is glucose autoxidation, and the glyoxal concentrations in plasma are higher in diabetic patients than in nondiabetics. Glyoxal reacts with biomolecules forming covalently modified DNA and protein adducts. We previously developed sensitive and specific assays based on nanoflow liquid chromatography-nanospray ionization tandem mass spectrometry (nanoLC-NSI/MS/MS) for quantification of DNA cross-linked adducts (dG-gx-dC and dG-gx-dA) and for hemoglobin adducts derived from glyoxal. In this study, we isolated and analyzed both leukocyte DNA and hemoglobin from the blood of diabetic patients and compared the adduct levels with those from nondiabetic subjects using the modified assays. The results indicated that the extents of glyoxal-induced hemoglobin modifications on α-Lys-11, α-Arg-92, β-Lys-17, and β-Lys-66 were statistically higher in diabetic patients than nondiabetics and they correlated with HbA1c significantly. Moreover, the levels of dG-gx-dC in leukocyte DNA correlated positively with the extents of globin modification at α-Lys-11 and β-Lys-17, while levels of dG-gx-dA correlated with those at α-Lys-11 and α-Arg-92 in nonsmoking subjects. Comparing the levels and the correlation coefficients of these hemoglobin and DNA adducts including or excluding smokers, it appears that smoking is not a major contributor to glyoxal-induced adduction of hemoglobin and leukocyte DNA. To the best of our knowledge, this is one of the few reports of positive correlation between DNA and protein adducts of the same compound (glyoxal) in the blood from the same subjects. Because of the high abundance of hemoglobin in blood, the results indicate that quantification of glyoxal-modified peptides in hemoglobin might serve as a dosimetry for glyoxal and a practical surrogate biomarker for assessing glyoxal-induced DNA damage and its prevention.

Malondialdehyde-Induced Post-translational Modifications in Hemoglobin of Smokers by NanoLC–NSI/MS/MS Analysis

“…Furthermore, hemoglobin can be obtained in pure form for analysis, so that interference from other proteins is minimal. Most importantly, levels of hemoglobin adducts are strongly correlated with DNA adducts of the same reactive species, as demonstrated in the cases of glyoxal, styrene, and 4-aminobiphenyl. − …”

Quantitation of Nitration, Chlorination, and Oxidation in Hemoglobin of Breast Cancer Patients by Nanoflow Liquid Chromatography Tandem Mass Spectrometry

“…The level of hemoglobin modification can provide a measure of exposure averaged in a 2-month period. ,− It has been demonstrated that the degree of DNA adduction in blood leukocytes is strongly correlated with that of hemoglobin modifications from the same blood sample. Therefore, measuring the extent of hemoglobin modification in peripheral blood can serve as a surrogate to monitor DNA damage for the assessment of cancer risk . Hemoglobin can be conveniently obtained from a dried blood spot (DBS) card, a widely used biomonitoring technique .…”

“…Therefore, measuring the extent of hemoglobin modification in peripheral blood can serve as a surrogate to monitor DNA damage for the assessment of cancer risk. 52 Hemoglobin can be conveniently obtained from a dried blood spot (DBS) card, a widely used biomonitoring technique. 53 We demonstrated that tyrosine nitration, methionine oxidation, and other types of hemoglobin modifications (from glyoxal and methylglyoxal) are stable in the DBS card stored at room temperature for a minimum of 2 weeks.…”

Analysis of Oxidative and Advanced Oxidative Modifications in Hemoglobin of Oral Cancer Patients by Mass Spectrometry

“…Hemoglobin adducts have been considered as surrogate biomarkers in biomonitoring the exposure of chemicals and carcinogens in vivo. − It is mainly owing to the high abundance of hemoglobin in the blood and its easy accessibility. In addition, the predictable lifetime of human hemoglobin and the lack of repair for protein adducts allow it to monitor the exposure of an individual on an average of 2 months. ,, …”

“…In this laboratory, we have identified various post-translational modifications (PTMs) of hemoglobin in human blood, including methionine and cysteine oxidation, glutathionylation of cysteine, chlorination, nitrosylation, and nitration of tyrosine, and carboxymethylation, carboxyethylation, and hydroimidazolone of lysine and arginine residues. ,− In addition, methylation and ethylation sites were identified on the N -termini, histidine, lysine, cysteine, and glutamate residues. , The extents of modification with specific types and sites were quantified relative to the native reference peptide (NRP) in the tryptic digest of hemoglobin. Among them, the extent of certain nitrotyrosine, glutathionylated cysteine, and ethylated residues are greater in smokers compared to nonsmokers, ,, while chlorotyrosine and glyoxal- and methylglyoxal-induced modification are elevated in diabetic patients. ,, …”