Measurement of Glutathionylated Haemoglobin by MAL-DI-ToF Mass Spectrometry as a Biomarker of Oxidative Stress in Heavy Smokers and in Occupational Obese Subjects

Abstract

Glutathionyl-haemoglobin (Hb-SSG) is a minor form of haemoglobin characterized by the presence of a disulfide bond between the β-93 cysteine residue and the thiol group of glutathione. Hb-SSG is naturally present in the erythrocytes at levels comparable to those of glycated haemoglobin and can be measured by MALDI mass spectrometry on very small samples of erythrocytes from peripheral blood. Since Hb-SSG has been recognized as a sensitive biomarker of oxidative stress in several degenerative diseases (diabetes, hyperlipidemia, kidney disease) and in healthy workers exposed to glutathione-depleting toxic agents such as butadiene, we have measured for the first time the levels of Hb-SSG in two groups: healthy heavy cigarette smokers and overweight-obese. For both classes of subjects, the measured levels (6.4% ± 1.7%, n = 30 for smokers; 3.0% ± 0.8%, n = 20 for overweight-obese) are in the upper 97 th percentile of those measured in the Italian general population. Levels in smokers show a small, yet statistically significant dependence on the level of smoking addiction (>20 cig./day vs. 20 cig./day: 7.0% ± 1.4% vs. 5.7% ± 1.1%; p < 0.05). This biomarker thus adds to those presently available to rationally assess the extent of biological damage caused by tobacco smoking.

Oxidative Stress Markers to Investigate the Effects of Hyperoxia in Anesthesia

“…As shown in panel b, most patients showed a high, but constant, level of HbSSG, expressed as a percentage of the total β Hb chain, before and throughout the surgery, with little influence from the FiO 2 level, except at the end of surgery, when they were higher in the FiO 2 = 0.80 group. Overall values were higher than those reported in the literature for healthy subjects [20].…”

Differential Redox State and Iron Regulation in Chronic Obstructive Pulmonary Disease, Acute Respiratory Distress Syndrome and Coronavirus Disease 2019

“…In the RBCs, glutathione occurs in two forms, either soluble ( Figure 4 , left panel) or bound to Hb as glutathione-Hb ( Figure 4 , middle panel). Glutathionyl Hb represents a minor form of Hb that indicates redox imbalance [ 26 , 28 ]. The right panel shows the total glutathione pool as the sum of the two forms.…”

“…For the MALDI analysis, a 10 µL sample was mixed to an equal volume of freshly prepared sinapinic acid matrix (Sigma-Aldrich, MALDI-grade brand, 30 mg/mL in 50% v / v acetonitrile—0.1% trifluoroacetic acid). Four one-microliter aliquots were manually spotted in adjacent circular wells of a stainless-steel plate, air-dried at room temperature, and loaded into the Bruker Autoflex III mass spectrometer for measurement as described [ 26 ]. Peak areas were measured with a custom spreadsheet, and glutathionylated-to-free thiol HbSSG/HbSH was expressed as a percent ratio.…”

The Redox Potential of the β-93-Cysteine Thiol Group in Human Hemoglobin Estimated from In Vitro Oxidant Challenge Experiments

“…Chronic rather than transient exposure to chemical oxidants occurs in diseases or conditions that impair the functioning of the enzymes involved in energy metabolism and in redox homeostasis. Normal, healthy subjects as well may face the consequences of exposure to an overload of oxidants, as the consequence of noxious lifestyles, such as tobacco smoking [29] and unhealthy dietary lifestyles [29]. Working under hazardous exposure to butadiene in a petrochemical plant [30] generates a high level of HbSSG in the workers.…”

“…Several clinical studies [18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33] report increased levels of HbSSG in etiologically diverse conditions that include: kidney impairment [18][19][20]; degenerative neurological and psychiatric diseases and treatments (Down's syndrome [21], Friedreich's ataxia [22], major depressive disorder [23], epilepsy [24]); diabetes [25][26][27][28]; hyperlipidemia [26]; obesity [29]; iron deficiency anemia [30]; the consequences of tobacco smoking [29]; and the exposure of petrochemical workers to butadiene, a carcinogenic airborne toxicant [31]. Some patients undergoing surgery with high-oxygen ventilation showed very high levels of HbSSG [32].…”