Journal: Transfusion

Article Title: Nicotine exposure increases markers of oxidant stress in stored red blood cells from healthy donor volunteers

doi: 10.1111/trf.15812

Figure Lengend Snippet: Metabolomics analysis of recalled REDS III donors ranked by cotinine levels, as measured by UHPLC–MS. The 5th and 95th percentile lowest and highest hemolyzers from the REDS III original cohort (n = 13,800) were asked to donate a second unit of blood, which was sampled at Storage Days 10, 23, and 42 for metabolomics analyses (A). Measurements of nicotine and its metabolites cotinine and cotinine oxide were obtained against deuterium-labeled internal standards (B). Cotinine levels were used to discriminate between smokers (or other nicotine exposures resulting in cotinine levels higher than 10 ng/mL – group indicated as “high” and represented as red dots in panel B) and nonsmokers (“low,” blue dots). In the same panel, cotinine, nicotine, cotinine oxide, and oxidative hemolysis measurements are shown for the subjects with low/blue versus high/red cotinine (B). Unsupervised principal component analysis (C), hierarchical clustering analysis (D), and pathway analyses (E) were performed on subjects characterized by low (blue) vs high (red) cotinine levels at Storage Days 10, 23, and 42, highlighting differences in energy and redox metabolism between the two groups at the freshest available storage time point (here Day 10) and at the end of storage. In E, since the color code may be too difficult to appreciate, numbers have been added to highlight the pathways that are most significantly affected between subjects with high and low levels of cotinine.

Article Snippet: 9 , 52 A 20-μL aliquot of each extract was treated with 2 μL of a mixture of deuterium (D3)-labeled internal standards for cotinine (DLM-1819-0.01, Cambridge Isotope Laboratories), nicotine (DLM-1818-PK, Cambridge Isotope Laboratories), and cotinine oxide (sc-219705, Santa Cruz Bio-technology) so that the final concentrations of the standards were 0.2 μM, then dried and resuspended in 22 μL of 0.1% formic acid in water.

Techniques: Labeling

Journal: Transfusion

Article Title: Nicotine exposure increases markers of oxidant stress in stored red blood cells from healthy donor volunteers

doi: 10.1111/trf.15812

Figure Lengend Snippet: Comparison of the metabolic phenotypes of the 15 subjects with the highest (red) and lowest (blue) levels of cotinine in the recalled REDS III donor population. A subgroup of the REDS III recalled donor population was identified on the basis of the highest (red) or lowest (blue) cotinine measurements at Day 10 (A) (n = 15 per group, n = 220 for the rest of the population with intermediate cotinine measurements in light gray). Highlights of the measurements of nicotine and its metabolites cotinine and cotinine oxide were obtained against deuterium-labeled internal standards in the 15 subjects with the highest or lowest cotinine levels at Day 10, along with the propensity of RBCs to hemolyze following oxidative insult with 2,2’-Azobis(2-amidinopropane) dihydrochloride (AAPH) (B). Unsupervised principal component analysis (C), hierarchical clustering analysis (D), and pathway analyses (E) were performed on the 15 subjects with the lowest levels of cotinine (indicating minimal exposure to smoke or other source of nicotine exposure [blue]) versus highest levels of cotinine (red) at Storage Days 10, 23, and 42, highlighting differences in energy and redox metabolism between the two groups at the freshest available storage time point (here Day 10) and at the end of storage.

Article Snippet: 9 , 52 A 20-μL aliquot of each extract was treated with 2 μL of a mixture of deuterium (D3)-labeled internal standards for cotinine (DLM-1819-0.01, Cambridge Isotope Laboratories), nicotine (DLM-1818-PK, Cambridge Isotope Laboratories), and cotinine oxide (sc-219705, Santa Cruz Bio-technology) so that the final concentrations of the standards were 0.2 μM, then dried and resuspended in 22 μL of 0.1% formic acid in water.

Techniques: Comparison, Labeling

Journal: Transfusion

Article Title: Nicotine exposure increases markers of oxidant stress in stored red blood cells from healthy donor volunteers

doi: 10.1111/trf.15812

Figure Lengend Snippet: The effect of nicotine exposure on RBC glycolysis (A) and PPP (B) in the 15 subjects with the highest (red) and lowest (blue) levels of cotinine in the recalled REDS III donor population. Red arrows highlight the subjects with the highest levels of cotinine when the subject is an outlier for a given metabolite measurement.

Article Snippet: 9 , 52 A 20-μL aliquot of each extract was treated with 2 μL of a mixture of deuterium (D3)-labeled internal standards for cotinine (DLM-1819-0.01, Cambridge Isotope Laboratories), nicotine (DLM-1818-PK, Cambridge Isotope Laboratories), and cotinine oxide (sc-219705, Santa Cruz Bio-technology) so that the final concentrations of the standards were 0.2 μM, then dried and resuspended in 22 μL of 0.1% formic acid in water.

Techniques:

Journal: Transfusion

Article Title: Nicotine exposure increases markers of oxidant stress in stored red blood cells from healthy donor volunteers

doi: 10.1111/trf.15812

Figure Lengend Snippet: The effect of nicotine exposure on RBC glutathione/ascorbate metabolism (A) and purine oxidation (B) in the 15 subjects with the highest (red) and lowest (blue) levels of cotinine in the recalled REDS III donor population. Red arrows highlight the subjects with the highest levels of cotinine when the subject is an outlier for a given metabolite measurement.

Article Snippet: 9 , 52 A 20-μL aliquot of each extract was treated with 2 μL of a mixture of deuterium (D3)-labeled internal standards for cotinine (DLM-1819-0.01, Cambridge Isotope Laboratories), nicotine (DLM-1818-PK, Cambridge Isotope Laboratories), and cotinine oxide (sc-219705, Santa Cruz Bio-technology) so that the final concentrations of the standards were 0.2 μM, then dried and resuspended in 22 μL of 0.1% formic acid in water.

Techniques:

Journal: Transfusion

Article Title: Nicotine exposure increases markers of oxidant stress in stored red blood cells from healthy donor volunteers

doi: 10.1111/trf.15812

Figure Lengend Snippet: The effect of nicotine exposure on RBC methionine (A) and lipid oxidation metabolism (B) in the 15 subjects with the highest (red) and lowest (blue) levels of cotinine in the recalled REDS III donor population. Red arrows highlight the subjects with the highest levels of cotinine when the subject is an outlier for a given metabolite measurement.

Article Snippet: 9 , 52 A 20-μL aliquot of each extract was treated with 2 μL of a mixture of deuterium (D3)-labeled internal standards for cotinine (DLM-1819-0.01, Cambridge Isotope Laboratories), nicotine (DLM-1818-PK, Cambridge Isotope Laboratories), and cotinine oxide (sc-219705, Santa Cruz Bio-technology) so that the final concentrations of the standards were 0.2 μM, then dried and resuspended in 22 μL of 0.1% formic acid in water.

Techniques:

Journal: Transfusion

Article Title: Nicotine exposure increases markers of oxidant stress in stored red blood cells from healthy donor volunteers

doi: 10.1111/trf.15812

Figure Lengend Snippet: Metabolic correlates to cotinine levels in the REDS III recalled donor population (A) and in the 15 subjects with the highest and lowest levels of cotinine (B). In C, correlation between relative and absolute cotinine levels (arbitrary units—[AU] vs. concentration—μM against stable isotope-labeled internal standards) in the REDS III RBC Omics recalled donor population. In D and E, positive correlates to cotinine measurements, including cotinine oxide in the whole population (D) or the 15 subjects with extreme highest or lowest cotinine measurements (E), nicotine, ferric gluconate, and anthranilate. In F, top negative correlates to cotinine. In G and H, correlations between cotinine and cotinine oxide and the smoking group defined by the self-reported questionnaire.

Article Snippet: 9 , 52 A 20-μL aliquot of each extract was treated with 2 μL of a mixture of deuterium (D3)-labeled internal standards for cotinine (DLM-1819-0.01, Cambridge Isotope Laboratories), nicotine (DLM-1818-PK, Cambridge Isotope Laboratories), and cotinine oxide (sc-219705, Santa Cruz Bio-technology) so that the final concentrations of the standards were 0.2 μM, then dried and resuspended in 22 μL of 0.1% formic acid in water.

Techniques: Concentration Assay, Labeling